Antibodies recognizing tau

ABSTRACT

The invention provides antibodies that specifically bind tau. The antibodies inhibit or delay tau-associated pathologies and associated symptomatic deterioration.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/091,060 filed Oct. 3, 2018, which is a national stage entry ofPCT/IB2017/052544 filed May 2, 2017, which claims the benefit of U.S.Provisional Application No. 62/330,789 filed May 2, 2016, each of whichis incorporated by reference in its entirety for all purposes.

REFERENCE TO A SEQUENCE LISTING

The Sequence Listing written in file 696318SEQLST.txt is 67,558 bytes,created on Nov. 3, 2020, and is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Tau is a well-known human protein that can exist in phosphorylated forms(see, e.g., Goedert, Proc. Natl. Acad. Sci. U.S.A. 85:4051-4055 (1988);Goedert, EMBO J. 8:393-399 (1989); Lee, Neuron 2:1615-1624 (1989);Goedert, Neuron 3:519-526 (1989); Andreadis, Biochemistry 31:10626-10633(1992). Tau has been reported to have a role in stabilizingmicrotubules, particularly in the central nervous system. Total tau(t-tau, i.e., phosphorylated and unphosphorylated forms) and phospho-tau(p-tau, i.e., phosphorylated tau) are released by the brain in responseto neuronal injury and neurodegeneration and have been reported to occurat increased levels in the CSF of Alzheimer's patients relative to thegeneral population (Jack et al., Lancet Neurol 9: 119-28 (2010)).

Tau is the principal constituent of neurofibrillary tangles, whichtogether with plaques are a hallmark characteristic of Alzheimer'sdisease. The tangles constitute abnormal fibrils measuring 10 nm indiameter occurring in pairs wound in a helical fashion with a regularperiodicity of 80 nm. The tau within neurofibrillary tangles isabnormally phosphorylated (hyperphosphorylated) with phosphate groupsattached to specific sites on the molecule. Severe involvement ofneurofibrillary tangles is seen in the layer II neurons of theentorhinal cortex, the CA1 and subicular regions of the hippocampus, theamygdala, and the deeper layers (layers III, V, and superficial VI) ofthe neocortex in Alzheimer's disease. Hyperphosphorylated tau has alsobeen reported to interfere with microtubule assembly, which may promoteneuronal network breakdown.

Tau inclusions are part of the defining neurophathology of severalneurodegenerative diseases including Alzheimer's disease, frontotemporallobar degeneration, progressive supranuclear palsy and Pick's disease.

BRIEF SUMMARY OF THE CLAIMED INVENTION

In one aspect, the invention provides an isolated monoclonal antibodythat specifically binds to tau. Examples of such antibodies bind to anepitope within amino acid residues 199-213 or 262-276 of SEQ ID NO:3(corresponding to amino acid residues 257-271 or 320-334, respectively,of SEQ ID NO:1).

Some such antibodies compete for binding to human tau with antibody 3D6.Some such antibodies bind to the same epitope on human tau as 3D6.

Some antibodies comprise three light chain CDRs as and three heavy chainCDRs of monoclonal antibody 3D6, wherein 3D6 is a mouse antibodycharacterized by a heavy chain variable region having an amino acidsequence comprising SEQ ID NO: 7 and a light chain variable regionhaving an amino acid sequence comprising SEQ ID NO: 11. In someantibodies, the three heavy chain CDRs are as defined by Kabat/ChothiaComposite (SEQ ID NOs: 8, 9, and 10) and the three light chain CDRs areas defined by Kabat/Chothia Composite (SEQ ID NOs: 12, 13, and 14).

For example, the antibody can be 3D6 or a chimeric, veneered, orhumanized form thereof. In some such antibodies, the variable heavychain has ≥85% identity to human sequence. In some such antibodies, thevariable light chain has ≥85% identity to human sequence. In some suchantibodies, each of the variable heavy chain and variable light chainhas ≥85% identity to human germline sequence.

In some such antibodies, the mature heavy chain variable regioncomprises the three heavy chain CDRs are as defined by Kabat/ChothiaComposite (SEQ ID NOs: 8, 9, and 10) and the three light chain CDRs areas defined by Kabat/Chothia Composite (SEQ ID NOs: 12, 13, and 14);provided that position H27 is occupied by F or Y, H28 is occupied by Nor T, H29 is occupied by I or F, H30 is occupied by K or T, position H51is occupied by I or V, position H54 is occupied by N or D, position H60is occupied by D or A, H61 is occupied by P or E, and H102 is occupiedby F or Y. In some such antibodies, CDR-H1 has an amino acid sequencecomprising SEQ ID NO: 42. In some such antibodies, CDR-H1 has an aminoacid sequence comprising SEQ ID NO: 58. In some such antibodies, CDR-H1has an amino acid sequence comprising SEQ ID NO: 59. In some suchantibodies, CDR-H1 has an amino acid sequence comprising SEQ ID NO: 60.In some such antibodies, CDR-H2 has an amino acid sequence comprisingSEQ ID NO: 43. In some such antibodies, CDR-H2 has an amino acidsequence comprising SEQ ID NO: 61. In some such antibodies, CDR-H2 hasan amino acid sequence comprising SEQ ID NO: 62. In some suchantibodies, CDR-H2 has an amino acid sequence comprising SEQ ID NO: 63.In some such antibodies, CDR-H2 has an amino acid sequence comprisingSEQ ID NO: 64. In some such antibodies, CDR-H3 has an amino acidsequence comprising SEQ ID NO: 65.

In some such antibodies, the antibody is a humanized antibody. In somesuch antibodies, CDR-H1 has an amino acid sequence comprising SEQ ID NO:42 and CDR-H2 has an amino acid sequence comprising SEQ ID NO: 43. Insome such antibodies, CDR-H1 has an amino acid sequence comprising SEQID NO: 42 and CDR-H2 has an amino acid sequence comprising SEQ ID NO:61.In some such antibodies, CDR-H1 has an amino acid sequence comprisingSEQ ID NO: 42 and CDR-H2 has an amino acid sequence comprising SEQ IDNO:64. In some such antibodies, CDR-H1 has an amino acid sequencecomprising SEQ ID NO: 42, CDR-H2 has an amino acid sequence comprisingSEQ ID NO:63, and CDR-H3 has an amino acid sequence comprising SEQ IDNO:65. In some such antibodies, CDR-H1 has an amino acid sequencecomprising SEQ ID NO: 58 and CDR-H2 has an amino acid sequencecomprising SEQ ID NO:62. In some such antibodies, CDR-H1 has an aminoacid sequence comprising SEQ ID NO: 59 and CDR-H2 has an amino acidsequence comprising SEQ ID NO:63. In some such antibodies, CDR-H1 has anamino acid sequence comprising SEQ ID NO: 60 and CDR-H2 has an aminoacid sequence comprising SEQ ID NO:62.

Some antibodies are a humanized or chimeric 3D6 antibody thatspecifically binds to human tau, wherein 3D6 is a mouse antibodycharacterized by a mature heavy chain variable region of SEQ ID NO:7 anda mature light chain variable region of SEQ ID NO: 11. Some suchantibodies are a humanized antibody comprising a humanized mature heavychain variable region comprising the three heavy chain CDRs of 3D6 and ahumanized mature light chain variable region comprising the three lightchain CDRs of 3D6. In some such antibodies, the CDRs are of a definitionselected from the group of Kabat, Chothia, Kabat/Chothia Composite, AbMand Contact.

In some such antibodies the humanized mature heavy chain variable regioncomprises the three Kabat/Chothia Composite heavy chain CDRs of 3D6 (SEQID NOs: 8-10) and the humanized mature light chain variable regioncomprises the three Kabat/Chothia Composite light chain CDRs of 3D6 (SEQID NOs: 12-14).

In some such antibodies, the humanized mature heavy chain variableregion comprises the three Kabat heavy chain CDRs of 3D6 (SEQ ID NO:32,SEQ ID NO:9, and SEQ ID NO:10) and the humanized mature light chainvariable region comprises the three Kabat light chain CDRs of 3D6 (SEQID NOs: 12-14).

In some such antibodies, the humanized mature heavy chain variableregion comprises the three Chothia heavy chain CDRs of 3D6 (SEQ IDNO:33, SEQ ID NO:34, and SEQ ID NO:10) and the humanized mature lightchain variable region comprises the three Chothia light chain CDRs of3D6 (SEQ ID NOs: 12-14).

In some such antibodies, the humanized mature heavy chain variableregion comprises the three AbM heavy chain CDRs of 3D6 (SEQ ID NO:8, SEQID NO:35, and SEQ ID NO:10)) and the humanized mature light chainvariable region comprises the three AbM light chain CDRs of 3D6 (SEQ IDNOs: 12-14).

In some such antibodies, the humanized mature heavy chain variableregion comprises the three Contact heavy chain CDRs of 3D6 (SEQ IDNOs:39-41) and the humanized mature light chain variable regioncomprises the three Contact light chain CDRs of 3D6 (SEQ ID NOs:36-38).

In some antibodies, the humanized mature heavy chain variable region hasan amino acid sequence at least 90% identical to any one of SEQ IDNOs:15-19 and SEQ ID NOs:46-57 and a humanized mature light chainvariable region having an amino acid sequence at least 90% identical toany one of SEQ ID NOs: 20-23, except that position H17 can be T or S,and position H20 can be I or V.

In some such antibodies, at least one of the following positions in theVH region is occupied by the amino acid as specified: H38 is occupied byR and H93 is occupied by S. In some such antibodies, positions H38 andH93 in the VH region are occupied by R and S, respectively.

In some such antibodies, at least one of the following positions in theVH region is occupied by the amino acid as specified: H38 is occupied byR, H43 is occupied by Q, H83 is occupied by T, and H93 is occupied by S.In some such antibodies, positions H38, H43, H83, and H93 in the VHregion are occupied by R, Q, T, and S, respectively.

In some antibodies, at least one of the following positions in the VHregion is occupied by the amino acid as specified: H12 is occupied by V,H24 is occupied by A, H48 is occupied by I, H67 is occupied by A, H80 isoccupied by L, H81 is occupied by Q, and H91 is occupied by F. In someantibodies, positions H12, H24, H48, H67, H80, H81, and H91 in the VHregion are occupied by V, A, I, A, L, Q, and F, respectively.

In some antibodies at least one of the following positions in the VHregion is occupied by the amino acid as specified: H13 is occupied by Rand H66 is occupied by K. In some antibodies, positions H13 and H66 inthe VH region are occupied by R and K, respectively.

In some antibodies, at least one of the following positions in the VHregion is occupied by the amino acid as specified: H40 is occupied by Rand H82a is occupied by G. In some antibodies, positions H40 and H82a inthe VH region are occupied by R and G, respectively.

In some antibodies, at least one of the following positions in the VHregion is occupied by the amino acid as specified: H42 is occupied by Eand H76 is occupied by N. In some antibodies, positions H42 and H76 inthe VH region are occupied by E and N, respectively.

In some antibodies, at least one of the following positions in the VHregion is occupied by the amino acid as specified: H40 is occupied by R,H82a is occupied by G, and H83 is occupied by T. In some antibodies,positions H40, H82a, and H83 in the VH region are occupied by R, G, andT, respectively.

In some antibodies, position H12 in the VH region is occupied by V.

In some antibodies, position H80 in the VH region is occupied by L.

In some antibodies, at least one of the following positions in the VHregion is occupied by the amino acid as specified: H24 is occupied by A,H48 is occupied by I, H67 is occupied by A, H80 is occupied by L, andH91 is occupied by F. In some antibodies, positions H24, H48, H67, H80,and H91 in the VH region are occupied by A, I, A, L, and F,respectively.

In some antibodies, at least one of the following positions in the VHregion is occupied by the amino acid as specified: H43 is occupied by Q,and H81 is occupied by Q. In some antibodies, positions H43 and H81 inthe VH region are occupied by Q, and Q, respectively.

In some antibodies, at least one of the following positions in the VHregion is occupied by the amino acid as specified: H24 is occupied by A,and H91 is occupied by F. In some antibodies, positions H24 and H91 inthe VH region are occupied by A and F, respectively.

In some antibodies, at least one of the following positions in the VHregion is occupied by the amino acid as specified: H13 is occupied by R,H17 is occupied by L, H29 is occupied by F, H42 is occupied by E, H43 isoccupied by Q, H61 is occupied by E, H76 is occupied by N, H80 isoccupied by L, H81 is occupied by Q. In some antibodies, positions H13,H17, H29, H42, H43, H61, H76, H80, and H81 in the VH region are occupiedby R, L, F, E, Q, E, N, L, and Q, respectively.

In some antibodies, at least one of the following positions in the VHregion is occupied by the amino acid as specified: H24 is occupied by A,H28 is occupied by T, H48 is occupied by I, H54 is occupied by D, H60 isoccupied by A, H67 is occupied by A, H80 is occupied by L, and H91 isoccupied by F. In some antibodies, positions H24, H28, H48, H54, H60,H67, H80, and H91 in the VH region are occupied by A, T, I, D, A, A, L,and F, respectively.

In some antibodies, at least one of the following positions in the VHregion is occupied by the amino acid as specified: H10 is occupied by D,H17 is occupied by L, H24 is occupied by A, H28 is occupied by T, H43 isoccupied by Q, H48 is occupied by I, H60 is occupied by A, H61 isoccupied by E, H91 is occupied by F, H108 is occupied by T, and H109 isoccupied by L. In some antibodies, positions H10, H17, H24, H28, H43,H48, H60, H61, H91, H108, and H109 in the VH region are occupied by D,L, A, T, Q, I, A, E, F, T, and L, respectively.

In some antibodies, at least one of the following positions in the VHregion is occupied by the amino acid as specified: H17 is occupied by L,H27 is occupied by Y, H29 is occupied by F, and H61 is occupied by E. Insome antibodies, positions H17, H27, H29, and H61 in the VH region areoccupied by L, Y, F, and E, respectively.

In some antibodies, at least one of the following positions in the VHregion is occupied by the amino acid as specified: H17 is occupied by L,H27 is occupied by Y, H29 is occupied by F, H61 is occupied by E, H76 isoccupied by N, and H82a is occupied by G. In some antibodies, positionsH17, H27, H29, H61, H76, and H82a in the VH region are occupied by L, Y,F, E, N, and G, respectively.

In some antibodies, at least one of the following positions in the VHregion is occupied by the amino acid as specified: H12 is occupied by V,H17 is occupied by L, H24 is occupied by A, H43 is occupied by Q, H48 isoccupied by I, H83 is occupied by T, and H91 is occupied by F. In someantibodies, positions H12, H17, H24, H43, H48, H83, and H91 in the VHregion are occupied by V, L, A, Q, I, T, F, respectively.

In some antibodies, at least one of the following positions in the VHregion is occupied by the amino acid as specified: H12 is occupied by V,H24 is occupied by A, H48 is occupied by I, H67 is occupied b A, H80 isoccupied by L, H83 is occupied by T, and H91 is occupied by F. In someantibodies, positions H12, H24, H48, H67, H80, H83, and H91 in the VHregion are occupied by V, A, I, A, L, T, and F, respectively.

In some antibodies, at least one of the following positions in the VHregion is occupied by the amino acid as specified: H10 is occupied by Eor D, H12 is occupied by K or V, H13 is occupied by K or R, H17 isoccupied by T, L or S, H24 is occupied by V or A, H27 is occupied by For Y, H28 is occupied by N or T, H29 is occupied by I or F, H30 isoccupied by K or T, H38 is occupied by Q or R, H40 is occupied by A orR, H42 is occupied by G or E, H43 is occupied by K or Q, H48 is occupiedby M or I, H51 is occupied by V or I, H54 is occupied by N or D, H60 isoccupied by D or A, H61 is occupied by P or E, H66 is occupied by R orK, H67 is occupied by V or A, H76 is occupied by D or N, H80 is occupiedby M or L, H81 is occupied by E or Q, H82a is occupied by S or G, H83 isoccupied by T or R, H91 is occupied by Y or F, H93 is occupied by A orS, H102 is occupied by F or Y, H108 is occupied by T or L, H109 isoccupied by L or V. In some antibodies, positions H12, H13, H17, H24,H38, H42, H43, H48, H66, H67, H76, H80, H81, H83, H91, and H93 in the VHregion are occupied by V, R, L, A, R, E, Q, I, K, A, N, L, Q, T, F, andS, respectively.

In some antibodies, positions H38, H42, H43, H76, H83, and H93 in the VHregion are occupied by R, E, Q, N, T, and S, respectively. In someantibodies, positions H12, H13, H17, H24, H38, H40, H42, H43, H48, H66,H67, H76, H80, H81, H82A, H83, H91, and H93 in the VH region areoccupied by V, R, L, A, R, R, E, Q, I, K, A, N, L, Q, G, T, F, and S,respectively. In some antibodies, positions H12, H24, H38, H40, H43,H48, H67, H80, H81, H82A, H83, H91, and H93 in the VH region areoccupied by V, A, R, R, Q, I, A, L, Q, G, T, F, and S, respectively. Insome antibodies, positions H12, H24, H28, H38, H40, H43, H48, H54, H60,H67, H80, H81, H82A, H83, H91, and H93 in the VH region are occupied byV, A, T, R, R, Q, I, D, A, A, L, Q, G, T, F, and S, respectively.

In some antibodies, positions H12, H24, H28, H38, H40, H48, H51, H54,H60, H67, H80, H82A, H83, H91, and H93 in the VH region are occupied byV, A, T, R, R, I, V, D, A, A, L, G, T, F, and S, respectively. In someantibodies, positions H12, H24, H28, H38, H40, H48, H54, H60, H67, H80,H82A, H83, H91, and H93 in the VH region are occupied by V, A, T, R, R,I, D, A, A, L, G, T, F, and S, respectively. In some antibodies,positions H13, H17, H24, H29, H38, H40, H42, H43, H54, H61, H76, H80,H81, H82A, H83, H91, and H93 in the VH region are occupied by R, L, A,F, R, R, E, Q, N, E, N, L, Q, G, T, F, and S, respectively.

In some antibodies, positions H13, H17, H24, H27, H28, H29, H30, H38,H40, H42, H43, H51, H54, H60, H61, H76, H80, H81, H82A, H83, H91, andH93 in the VH region are occupied by R, L, A, Y, T, F, T, R, R, E, Q, V,D, A, E, N, L, Q, G, T, F, and S, respectively. In some antibodies,positions H10, H12, H17, H24, H28, H38, H40, H42, H43, H48, H54, H60,H61, H76, H80, H82A, H83, H91, H93, H108, and H109 in the VH region areoccupied by D, V, L, A, T, R, R, E, Q, I, N, A, E, N, L, G, T, F, S, T,and L, respectively. In some antibodies, positions H10, H12, H17, H24,H28, H38, H40, H43, H48, H51, H54, H60, H61, H82A, H83, H91, H93, H102,H108, and H109 in the VH region are occupied by D, V, L, A, T, R, R, Q,I, V, D, A, E, G, T, F, S, Y, T, and L, respectively.

In some antibodies, positions H38 and H93 in the VH region are occupiedby R and S, respectively. In some antibodies, positions H17, H27, H29,H38, H61, H76, H82A, and H93 in the VH region are occupied by L, Y, F,R, E, N, G, and S, respectively. In some antibodies, positions H17, H27,H28, H29, H30, H38, H51, H54, H60, H61, H76, H82A, and H93 in the VHregion are occupied by L, Y, T, F, T, R, V, D, A, E, N, G, and S,respectively.

In some antibodies, positions H12, H38, H40, H48, H66, H67, H76, H80,H82A, H83, and H93 in the VH region are occupied by V, R, R, I, K, A, N,L, G, T, and S, respectively.

In some antibodies, positions H12, H17, H27, H29, H38, H40, H61, H80,H82A, H83, and H93 in the VH region are occupied by V, L, Y, F, R, R, E,L, G, T, and S, respectively. In some antibodies, positions H12, H17,H27, H28, H29, H30, H38, H40, H51, H54, H60, H61, H80, H82A, H83, andH93 in the VH region are occupied by V, L, Y, T, F, T, R, R, V, D, A, E,L, G, T, and S, respectively.

In some antibodies, at least one of the following positions in the VLregion is occupied by the amino acid as specified: L36 is occupied by L,L37 is occupied by L, and L100 is occupied by G. In some antibodies,positions L36, L37, and L100 in the VL region are occupied by L, L, andG, respectively.

In some antibodies, at least one of the following positions in the VLregion is occupied by the amino acid as specified: L12 is occupied by Sand L45 is occupied by K. In some antibodies, positions L12 and L45 inthe VL region are occupied by S and K, respectively.

In some antibodies, at least one of the following positions in the VLregion is occupied by the amino acid as specified: L2 is V or I, L7 is Sor T, L12 is P or S, L15 is L or I, L36 is L, L37 is L, L45 is R or K,L60 is D or S, L100 is G.

In some antibodies, positions L12, L36, L37, L45, and L100 in the VLregion are occupied by S, L, L, K, and G, respectively. In someantibodies, positions L36, L37, and L100 in the VL region are occupiedby L, L and G, respectively. In some antibodies, positions L36, L37,L60, and L100 in the VL region are occupied by L, L, S, and G,respectively. In some antibodies, positions L2, L7, L12, L15, L36, L37,L45, and L100 in the VL region are occupied by I, T, S, I, L, L, K, andG, respectively. In some antibodies, positions L2, L7, L12, L15, L36,L37, L45, and L100 in the VL region are occupied by V, S, P, L, L, L, R,and G, respectively.

Some antibodies comprise a mature heavy chain variable region having anamino acid sequence at least 95% identical to any one of SEQ ID NOs:15-19, 46-57 and a mature light chain variable region having an aminoacid sequence at least 95% identical to any one of SEQ ID NOs: 20-23,except that position H17 can be T or S, and position H20 can be I or V.Some antibodies comprise a mature heavy chain variable region having anamino acid sequence at least 98% identical to any one of SEQ ID NOs:15-19, 46-57 and a mature light chain variable region having an aminoacid sequence at least 98% identical to any one of SEQ ID NOs: 20-23,except that position H17 can be T or S, and position H20 can be I or V.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence of any one of SEQ ID NOs:15-19 and SEQ ID NOs:46-57 andthe mature light chain variable region has an amino acid sequence of anyone of SEQ ID NOs:20-23. In some antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:15 and themature light chain variable region has an amino acid sequence of SEQ IDNO:20. In some antibodies, the mature heavy chain variable region has anamino acid sequence of SEQ ID NO:15 and the mature light chain variableregion has an amino acid sequence of SEQ ID NO:21. In some antibodies,the mature heavy chain variable region has an amino acid sequence of SEQID NO:15 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:22. In some antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:15 and themature light chain variable region has an amino acid sequence of SEQ IDNO:23.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence of SEQ ID NO:16 and the mature light chain variable regionhas an amino acid sequence of SEQ ID NO:20. In some antibodies, themature heavy chain variable region has an amino acid sequence of SEQ IDNO:16 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:21. In some antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:16 and themature light chain variable region has an amino acid sequence of SEQ IDNO:22. In some antibodies, the mature heavy chain variable region has anamino acid sequence of SEQ ID NO:16 and the mature light chain variableregion has an amino acid sequence of SEQ ID NO:23.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence of SEQ ID NO:17 and the mature light chain variable regionhas an amino acid sequence of SEQ ID NO:20. In some antibodies, themature heavy chain variable region has an amino acid sequence of SEQ IDNO:17 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:21. In some antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:17 and themature light chain variable region has an amino acid sequence of SEQ IDNO:22. In some antibodies, the mature heavy chain variable region has anamino acid sequence of SEQ ID NO:17 and the mature light chain variableregion has an amino acid sequence of SEQ ID NO:23.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence of SEQ ID NO:18 and the mature light chain variable regionhas an amino acid sequence of SEQ ID NO:20. In some antibodies, themature heavy chain variable region has an amino acid sequence of SEQ IDNO:18 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:21. In some antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:18 the maturelight chain variable region has an amino acid sequence of SEQ ID NO:22.In some antibodies, the mature heavy chain variable region has an aminoacid sequence of SEQ ID NO:18 and the mature light chain variable regionhas an amino acid sequence of SEQ ID NO:23.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence of SEQ ID NO:19 and the mature light chain variable regionhas an amino acid sequence of SEQ ID NO:20. In some antibodies, themature heavy chain variable region has an amino acid sequence of SEQ IDNO:19 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:21. In some antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:19 and themature light chain variable region has an amino acid sequence of SEQ IDNO:22. In some antibodies, the mature heavy chain variable region has anamino acid sequence of SEQ ID NO:19 and the mature light chain variableregion has an amino acid sequence of SEQ ID NO:23.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence of SEQ ID NO:46 and the mature light chain variable regionhas an amino acid sequence of SEQ ID NO:20. In some antibodies, themature heavy chain variable region has an amino acid sequence of SEQ IDNO:46 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:21. In some antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:46 and themature light chain variable region has an amino acid sequence of SEQ IDNO:22. In some antibodies, the mature heavy chain variable region has anamino acid sequence of SEQ ID NO:46 and the mature light chain variableregion has an amino acid sequence of SEQ ID NO:23.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence of SEQ ID NO:47 and the mature light chain variable regionhas an amino acid sequence of SEQ ID NO:20. In some antibodies, themature heavy chain variable region has an amino acid sequence of SEQ IDNO:47 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:21. In some antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:47 and themature light chain variable region has an amino acid sequence of SEQ IDNO:22. In some antibodies, the mature heavy chain variable region has anamino acid sequence of SEQ ID NO:47 and the mature light chain variableregion has an amino acid sequence of SEQ ID NO:23.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence of SEQ ID NO:48 and the mature light chain variable regionhas an amino acid sequence of SEQ ID NO:20. In some antibodies, themature heavy chain variable region has an amino acid sequence of SEQ IDNO:48 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:21. In some antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:48 and themature light chain variable region has an amino acid sequence of SEQ IDNO:22. In some antibodies, the mature heavy chain variable region has anamino acid sequence of SEQ ID NO:48 and the mature light chain variableregion has an amino acid sequence of SEQ ID NO:23.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence of SEQ ID NO:49 and the mature light chain variable regionhas an amino acid sequence of SEQ ID NO:20. In some antibodies, themature heavy chain variable region has an amino acid sequence of SEQ IDNO:49 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:21. In some antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:49 and themature light chain variable region has an amino acid sequence of SEQ IDNO:22. In some antibodies, the mature heavy chain variable region has anamino acid sequence of SEQ ID NO:49 and the mature light chain variableregion has an amino acid sequence of SEQ ID NO:23.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence of SEQ ID NO:50 and the mature light chain variable regionhas an amino acid sequence of SEQ ID NO:20. In some antibodies, themature heavy chain variable region has an amino acid sequence of SEQ IDNO:50 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:21. In some antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:50 and themature light chain variable region has an amino acid sequence of SEQ IDNO:22. In some antibodies, the mature heavy chain variable region has anamino acid sequence of SEQ ID NO:50 and the mature light chain variableregion has an amino acid sequence of SEQ ID NO:23.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence of SEQ ID NO:51 and the mature light chain variable regionhas an amino acid sequence of SEQ ID NO:20. In some antibodies, themature heavy chain variable region has an amino acid sequence of SEQ IDNO:51 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:21. In some antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:51 and themature light chain variable region has an amino acid sequence of SEQ IDNO:22. In some antibodies, the mature heavy chain variable region has anamino acid sequence of SEQ ID NO:51 and the mature light chain variableregion has an amino acid sequence of SEQ ID NO:23.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence of SEQ ID NO:52 and the mature light chain variable regionhas an amino acid sequence of SEQ ID NO:20. In some antibodies, themature heavy chain variable region has an amino acid sequence of SEQ IDNO:52 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:21. In some antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:52 and themature light chain variable region has an amino acid sequence of SEQ IDNO:22. In some antibodies, the mature heavy chain variable region has anamino acid sequence of SEQ ID NO:52 and the mature light chain variableregion has an amino acid sequence of SEQ ID NO:23.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence of SEQ ID NO:53 and the mature light chain variable regionhas an amino acid sequence of SEQ ID NO:20. In some antibodies, themature heavy chain variable region has an amino acid sequence of SEQ IDNO:53 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:21. In some antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:53 and themature light chain variable region has an amino acid sequence of SEQ IDNO:22. In some antibodies, the mature heavy chain variable region has anamino acid sequence of SEQ ID NO:53 and the mature light chain variableregion has an amino acid sequence of SEQ ID NO:23.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence of SEQ ID NO:54 and the mature light chain variable regionhas an amino acid sequence of SEQ ID NO:20. In some antibodies, themature heavy chain variable region has an amino acid sequence of SEQ IDNO:54 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:21. In some antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:54 and themature light chain variable region has an amino acid sequence of SEQ IDNO:22. In some antibodies, the mature heavy chain variable region has anamino acid sequence of SEQ ID NO:54 and the mature light chain variableregion has an amino acid sequence of SEQ ID NO:23.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence of SEQ ID NO:55 and the mature light chain variable regionhas an amino acid sequence of SEQ ID NO:20. In some antibodies, themature heavy chain variable region has an amino acid sequence of SEQ IDNO:55 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:21. In some antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:55 and themature light chain variable region has an amino acid sequence of SEQ IDNO:22. In some antibodies, the mature heavy chain variable region has anamino acid sequence of SEQ ID NO:55 and the mature light chain variableregion has an amino acid sequence of SEQ ID NO:23.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence of SEQ ID NO:56 and the mature light chain variable regionhas an amino acid sequence of SEQ ID NO:20. In some antibodies, themature heavy chain variable region has an amino acid sequence of SEQ IDNO:56 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:21. In some antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:56 and themature light chain variable region has an amino acid sequence of SEQ IDNO:22. In some antibodies, the mature heavy chain variable region has anamino acid sequence of SEQ ID NO:56 and the mature light chain variableregion has an amino acid sequence of SEQ ID NO:23.

In some antibodies, the mature heavy chain variable region has an aminoacid sequence of SEQ ID NO:57 and the mature light chain variable regionhas an amino acid sequence of SEQ ID NO:20. In some antibodies, themature heavy chain variable region has an amino acid sequence of SEQ IDNO:57 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:21. In some antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:57 and themature light chain variable region has an amino acid sequence of SEQ IDNO:22. In some antibodies, the mature heavy chain variable region has anamino acid sequence of SEQ ID NO:57 and the mature light chain variableregion has an amino acid sequence of SEQ ID NO:23.

For example, the antibody can be a chimeric antibody. For example, theantibody can be a veneered antibody.

The antibody can be an intact mouse, chimeric, veneered or humanizedantibody or a binding fragment, single-chain antibody Fab fragment,Fab′2 fragment, or single chain Fv. Some of the antibodies have a humanIgG1 isotype, while others may have a human IgG2 or IgG4 isotype. Someantibodies have the mature light chain variable region fused to a lightchain constant region and the mature heavy chain variable region fusedto a heavy chain constant region. The heavy chain constant region ofsome antibodies is a mutant form of a natural human heavy chain constantregion which has reduced binding to a Fcγ receptor relative to thenatural human heavy chain constant region.

Some antibodies may have at least one mutation in the constant region,such as a mutation that reduces complement fixation or activation by theconstant region, for example, a mutation at one or more of positions241, 264, 265, 270, 296, 297, 318, 320, 322, 329 and 331 by EUnumbering. Some antibodies have an alanine at positions 318, 320 and322. Some antibodies can be at least 95% w/w pure. The antibody can beconjugated to a therapeutic, cytotoxic, cytostatic, neurotrophic, orneuroprotective agent.

In another aspect, the invention provides a pharmaceutical compositioncomprising any of the antibodies disclosed herein and apharmaceutically-acceptable carrier.

In another aspect, the invention provides a nucleic acid encoding theheavy chain and/or light chain of any of the antibodies disclosedherein, a recombinant expression vector comprising the nucleic acid anda host cell transformed with the recombinant expression vector.

In yet another aspect, the invention provides methods of humanizing anynon-human antibody described herein, for example, mouse antibody 3D6,wherein 3D6 is characterized by a mature heavy chain variable region ofSEQ ID NO:7 and a mature light chain variable region of SEQ ID NO:11.Such methods can involve selecting one or more acceptor antibodies,synthesizing a nucleic acid encoding a humanized heavy chain comprisingCDRs of the mouse heavy chain and a nucleic acid encoding a humanizedlight chain comprising CDRs of the mouse antibody light chain, andexpressing the nucleic acids in a host cell to produce a humanizedantibody.

Methods of producing antibodies, such as a humanized, chimeric orveneered antibody, for example humanized, chimeric or veneered forms of3D6, are also provided. In such methods, cells transformed with nucleicacids encoding the heavy and light chains of the antibody are culturedso that the cells secrete the antibody. The antibody can then bepurified from the cell culture media.

Cell lines producing any of the antibodies disclosed herein can beproduced by introducing a vector encoding heavy and light chains of theantibody and a selectable marker into cells, propagating the cells underconditions to select for cells having increased copy number of thevector, isolating single cells from the selected cells; and bankingcells cloned from a single cell selected based on yield of antibody.

Some cells can be propagated under selective conditions and screened forcell lines naturally expressing and secreting at least 100 mg/L/10⁶cells/24 hours. Single cells can be isolated from the selected cells.Cells cloned from a single cell can then be banked. Single cells can beselected based on desirable properties, such as the yield of theantibody. Exemplary cell lines are cell lines expressing 3D6.

The invention also provides methods of inhibiting or reducingaggregation of tau in a subject having or at risk of developing atau-mediated amyloidosis, comprising administering to the subject aneffective regime of an antibody disclosed herein, thereby inhibiting orreducing aggregation of tau in the subject. Exemplary antibodies includehumanized versions of 3D6.

Also provided are methods of treating or effecting prophylaxis of atau-related disease in a subject, comprising administering an effectiveregime of an antibody disclosed herein and thereby treating or effectingprophylaxis of the disease. Examples of such a disease are Alzheimer'sdisease, Down's syndrome, mild cognitive impairment, primary age-relatedtauopathy, postencephalitic parkinsonism, posttraumatic dementia ordementia pugilistica, Pick's disease, type C Niemann-Pick disease,supranuclear palsy, frontotemporal dementia, frontotemporal lobardegeneration, argyrophilic grain disease, globular glial tauopathy,amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam,corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy bodyvariant of Alzheimer disease (LBVAD), or progressive supranuclear palsy(PSP). In some methods, the tau-related disease is Alzheimer's disease.In some methods the patient is an ApoE4 carrier.

Also provided are methods of reducing aberrant transmission of taucomprising administering an effective regime of an antibody disclosedherein and thereby reducing transmission of tau.

Also provided are methods of inducing phagocytosis of tau comprisingadministering an effective regime of an antibody disclosed herein andthereby inducing phagocytosis of tau.

Also provided are methods of inhibiting tau aggregation or depositioncomprising administering an effective regime of an antibody disclosedherein thereby inhibiting tau aggregation or deposition.

Also provided are methods of inhibiting formation of tau tanglescomprising administering an effective regime of an antibody disclosedherein.

The invention also provides a method of detecting tau protein depositsin a subject having or at risk of a disease associated with tauaggregation or deposition comprising administering to a subject anantibody disclosed herein, and detecting the antibody bound to tau inthe subject. Examples of such a disease are Alzheimer's disease, Down'ssyndrome, mild cognitive impairment, primary age-related tauopathy,postencephalitic parkinsonism, posttraumatic dementia or dementiapugilistica, Pick's disease, type C Niemann-Pick disease, supranuclearpalsy, frontotemporal dementia, frontotemporal lobar degeneration,argyrophilic grain disease, globular glial tauopathy, amyotrophiclateral sclerosis/parkinsonism dementia complex of Guam, corticobasaldegeneration (CBD), dementia with Lewy bodies, Lewy body variant ofAlzheimer disease (LBVAD), or progressive supranuclear palsy (PSP). Insome embodiments, the antibody is administered by intravenous injectioninto the body of the subject. In some embodiments, the antibody isadministered directly to the brain of the subject by intracranialinjection or by drilling a hole through the skull of the subject. Insome embodiments, the antibody is labeled. In some embodiments, theantibody is labeled with a fluorescent label, a paramagnetic label, or aradioactive label. In some embodiments, the radioactive label isdetected using positron emission tomography (PET) or single-photonemission computed tomography (SPECT).

The invention also provides a method of measuring efficacy of treatmentin a subject being treated for a disease associated with tau aggregationor deposition, comprising measuring a first level of tau proteindeposits in the subject prior to treatment by administering to a subjectan antibody disclosed herein, and detecting a first amount of theantibody bound to tau in the subject, administering the treatment to thesubject, measuring a second level of tau protein deposits in the insubject after treatment by administering to a subject the antibody, anddetecting the antibody bound to tau in the subject, wherein a decreasein the level of tau protein deposits indicates a positive response totreatment.

The invention also provides a method of measuring efficacy of treatmentin a subject being treated for a disease associated with tau aggregationor deposition, comprising measuring a first level of tau proteindeposits in the subject prior to treatment by administering to a subjectan antibody disclosed herein, and detecting a first amount of antibodybound to tau in the subject, administering the treatment to the subject,measuring a second level of tau protein deposits in the in subject aftertreatment by administering to a subject the antibody, and detecting asecond amount of antibody bound to tau in the subject, wherein no changein the level of tau protein deposits or a small increase in tau proteindeposits indicates a positive response to treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the results of experiments designed to map the epitope(s)bound by the murine 3D6 monoclonal antibody.

FIG. 2 depicts an alignment of heavy chain variable regions of the mouse3D6 antibody and humanized versions of the 3D6 antibody (VHv1, VHv2,VHv1b, VHv1bA11, and VHv5). The consensus amino acid sequence among theheavy chain variable regions of the mouse 3D6 antibody and humanizedversions of the 3D6 antibody (VHv1, VHv2, VHv1b, VHv1bA11, and VHv5) islabeled “Majority.’ The CDRs as defined by Kabat/Chothia Composite arein boldface. Positions in heavy chain variable regions of the mouse 3D6antibody and humanized versions of the 3D6 antibody (VHv1, VHv2, VHv1b,VHv1bA11, and VHv5) where amino acid residues differ from the “Majority”sequence are boxed.

FIG. 3 depicts an alignment of light chain variable regions of the mouse3D6 antibody and humanized versions of the 3D6 antibody. The consensusamino acid sequence between the light chain variable regions of themouse 3D6 antibody and selected humanized 3D6 antibodies is labeled“Majority.’ The CDRs as defined by Kabat are in boldface. Positions inlight chain variable regions of the mouse 3D6 antibody and humanizedversions of the 3D6 antibody where amino acid residues differ from the“Majority” sequence are boxed.

FIGS. 4A and 4B depict an alignment of heavy chain variable regions ofthe mouse 3D6 antibody and humanized versions of the 3D6 antibody (VHv1,VHv1b, VHv1bA11, VHv1bA11B6G2, VHv1bA11B6H3, VHv1c, VHv1d, VHv1e, VHv1f,VHv2, VHv3, VHv3b, VHv3c, VHv4, VHv4b, VHv4c, and VHv5). The consensusamino acid sequence among the heavy chain variable regions of thehumanized versions of the 3D6 antibody (VHv1, VHv1b, VHv1bA11,VHv1bA11B6G2, VHv1bA11B6H3, VHv1c, VHv1d, VHv1e, VHv1f, VHv2, VHv3,VHv3b, VHv3c, VHv4, VHv4b, VHv4c, and VHv5) is labeled “Majority.’ TheCDRs as defined by Kabat/Chothia Composite are in boldface. Positions inheavy chain variable regions of the humanized versions of the 3D6antibody (VHv1, VHv1b, VHv1bA11, VHv1bA11B6G2, VHv1bA11B6H3, VHv1c,VHv1d, VHv1e, VHv1f, VHv2, VHv3, VHv3b, VHv3c, VHv4, VHv4b, VHv4c, andVHv5) where amino acid residues differ from the “Majority” sequence areboxed.

FIGS. 5A, 5B, and 5C depict results of ELISA screening assays forselected mouse monoclonal anti-tau antibodies.

FIG. 6 depicts binding kinetics for selected mouse monoclonal anti-tauantibodies to recombinant human tau.

FIG. 7 depicts results of functional blocking assays for selected mousemonoclonal anti-tau antibodies.

FIG. 8 depicts results of disaggregation assays for selected mousemonoclonal anti-tau antibodies.

FIG. 9 depicts results of experiments showing that 3D6 and 5G8immunocapture tau from human Alzheimer's disease tissue.

FIGS. 10A, 10B, and 10C depict results of experiments showing affinityof humanized 3D6 variants toward tau.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 sets forth the amino acid sequence of an isoform of humantau (Swiss-Prot P10636-8).

SEQ ID NO:2 sets forth the amino acid sequence of an isoform of humantau (Swiss-Prot P10636-7).

SEQ ID NO:3 sets forth the amino acid sequence of an isoform of humantau (Swiss-Prot P10636-6), (4R0N human tau).

SEQ ID NO:4 sets forth the amino acid sequence of an isoform of humantau (Swiss-Prot P10636-5)

SEQ ID NO:5 sets forth the amino acid sequence of an isoform of humantau (Swiss-Prot P10636-4).

SEQ ID NO:6 sets forth the amino acid sequence of an isoform of humantau (Swiss-Prot P10636-2).

SEQ ID NO: 7 sets forth the amino acid sequence of the heavy chainvariable region of the mouse 3D6 antibody.

SEQ ID NO: 8 sets forth the amino acid sequence of Kabat/Chothiacomposite CDR-H1 of the mouse 3D6 antibody.

SEQ ID NO:9 sets forth the amino acid sequence of Kabat CDR-H2 of themouse 3D6 antibody.

SEQ ID NO: 10 sets forth the amino acid sequence of Kabat CDR-H3 of themouse 3D6 antibody.

SEQ ID NO: 11 sets forth the amino acid sequence of the light chainvariable region of the mouse 3D6 antibody and of the mouse 6A10antibody.

SEQ ID NO: 12 sets forth the amino acid sequence of Kabat CDR-L1 of themouse 3D6 antibody and of the mouse 6A10 antibody.

SEQ ID NO: 13 sets forth the amino acid sequence of Kabat CDR-L2 of themouse 3D6 antibody and of the mouse 6A10 antibody.

SEQ ID NO: 14 sets forth the amino acid sequence of Kabat CDR-L3 of themouse 3D6 antibody and of the mouse 6A10 antibody.

SEQ ID NO:15 sets forth the amino acid sequence of heavy chain variableregion of the humanized 3D6 antibody hu3D6VHv1.

SEQ ID NO:16 sets forth the amino acid sequence of heavy chain variableregion of the humanized 3D6 antibody hu3D6VHv2.

SEQ ID NO:17 sets forth the amino acid sequence of heavy chain variableregion of the humanized 3D6 antibody hu3D6VHv1b.

SEQ ID NO:18 sets forth the amino acid sequence of heavy chain variableregion of the humanized 3D6 antibody hu3D6VHv1bA11.

SEQ ID NO:19 sets forth the amino acid sequence of heavy chain variableregion of the humanized 3D6 antibody hu3D6VHv5:

SEQ ID NO:20 sets forth the amino acid sequence of the light chainvariable region of the humanized 3D6 antibody hu3D6VLv1.

SEQ ID NO:21 sets forth the amino acid sequence of the light chainvariable region of the humanized 3D6 antibody hu3D6VLv2.

SEQ ID NO:22 sets forth the amino acid sequence of the light chainvariable region of the humanized 3D6 antibody hu3D6VLv3.

SEQ ID NO:23 sets forth the amino acid sequence of the light chainvariable region of the humanized 3D6 antibody hu3D6VLv4.

SEQ ID NO:24 sets forth the amino acid sequence of the heavy chainvariable acceptor Acc. #BAC01986.1.

SEQ ID NO:25 sets forth the amino acid sequence of the heavy chainvariable acceptor Acc. #IMGT #IGHV1-69-2*01.

SEQ ID NO:26 sets forth the amino acid sequence of the heavy chainvariable acceptor Acc. #IMGT #IGKJ1*01.

SEQ ID NO:27 sets forth the amino acid sequence of the light chainvariable acceptor Acc. #IMGT #IGKV2-30*02

SEQ ID NO:28 sets forth the amino acid sequence of the light chainvariable acceptor Acc. #IMGT #IGKJ2*01.

SEQ ID NO:29 sets forth the amino acid sequence of the light chainvariable acceptor Acc. #AAZ09048.1.

SEQ ID NO: 30 sets forth a nucleic acid sequence encoding the heavychain variable region of the mouse 3D6 antibody.

SEQ ID NO: 31 sets forth a nucleic acid sequence encoding the lightchain variable region of the mouse 3D6 antibody.

SEQ ID NO: 32 sets forth the amino acid sequence of Kabat CDR-H1 of themouse 3D6 antibody.

SEQ ID NO: 33 sets forth the amino acid sequence of Chothia CDR-H1 ofthe mouse 3D6 antibody.

SEQ ID NO: 34 sets forth the amino acid sequence of Chothia CDR-H2 ofthe mouse 3D6 antibody.

SEQ ID NO: 35 sets forth the amino acid sequence of AbM CDR-H2 of themouse 3D6 antibody.

SEQ ID NO: 36 sets forth the amino acid sequence of Contact CDR-L1 ofthe mouse 3D6 antibody.

SEQ ID NO: 37 sets forth the amino acid sequence of Contact CDR-L2 ofthe mouse 3D6 antibody.

SEQ ID NO: 38 sets forth the amino acid sequence of Contact CDR-L3 ofthe mouse 3D6 antibody.

SEQ ID NO: 39 sets forth the amino acid sequence of Contact CDR-H1 ofthe mouse 3D6 antibody.

SEQ ID NO: 40 sets forth the amino acid sequence of Contact CDR-H2 ofthe mouse 3D6 antibody.

SEQ ID NO: 41 sets forth the amino acid sequence of Contact CDR-H3 ofthe mouse 3D6 antibody.

SEQ ID NO: 42 sets forth the amino acid sequence of an alternateKabat-Chothia Composite CDR-H1 of a humanized 3D6 antibody (derived fromhu3D6VHv5, hu3D6VHv1bA11B6G2, hu3D6VHv1bA11B6H3, hu3D6VHv1e, andhu3D6VHv1f).

SEQ ID NO: 43 sets forth the amino acid sequence of an alternate KabatCDR-H2 of a humanized 3D6 antibody (derived from hu3D6VHv5 andhu3D6VHv1bA11B6H3).

SEQ ID NO:44 sets forth the consensus amino acid sequence among theheavy chain variable regions of the mouse 3D6 and selected humanized 3D6antibodies (VHv1, VHv2, VHv1b, VHv1bA11, and VHv5) (labeled “Majority’in FIG. 2 ).

SEQ ID NO:45 sets forth the consensus amino acid sequence between thelight chain variable regions of the mouse 3D6 and selected humanized 3D6antibodies (labeled “Majority’ in FIG. 3 ).

SEQ ID NO:46 sets forth the amino acid sequence of heavy chain variableregion of the humanized 3D6 antibody hu3D6VHv1bA11B6G2.

SEQ ID NO:47 sets forth the amino acid sequence of heavy chain variableregion of the humanized 3D6 antibody hu3D6VHv1bA11B6H3.

SEQ ID NO:48 sets forth the amino acid sequence of heavy chain variableregion of the humanized 3D6 antibody hu3D6VHv1c.

SEQ ID NO:49 sets forth the amino acid sequence of heavy chain variableregion of the humanized 3D6 antibody hu3D6VHv1d.

SEQ ID NO:50 sets forth the amino acid sequence of heavy chain variableregion of the humanized 3D6 antibody hu3D6VHv1e.

SEQ ID NO:51 sets forth the amino acid sequence of heavy chain variableregion of the humanized 3D6 antibody hu3D6VHv1f.

SEQ ID NO:52 sets forth the amino acid sequence of heavy chain variableregion of the humanized 3D6 antibody hu3D6VHv3.

SEQ ID NO:53 sets forth the amino acid sequence of heavy chain variableregion of the humanized 3D6 antibody hu3D6VHv3b.

SEQ ID NO:54 sets forth the amino acid sequence of heavy chain variableregion of the humanized 3D6 antibody hu3D6VHv3c.

SEQ ID NO:55 sets forth the amino acid sequence of heavy chain variableregion of the humanized 3D6 antibody hu3D6VHv4.

SEQ ID NO:56 sets forth the amino acid sequence of heavy chain variableregion of the humanized 3D6 antibody hu3D6VHv4b.

SEQ ID NO:57 sets forth the amino acid sequence of heavy chain variableregion of the humanized 3D6 antibody hu3D6VHv4c.

SEQ ID NO: 58 sets forth the amino acid sequence of an alternateKabat-Chothia Composite CDR-H1 of a humanized 3D6 antibody (derived fromhu3D6VH1c).

SEQ ID NO: 59 sets forth the amino acid sequence of an alternateKabat-Chothia Composite CDR-H1 of a humanized 3D6 antibody (derived fromhu3D6VHv1d, hu3D6VHv3c, and hu3D6VHv4c).

SEQ ID NO: 60 sets forth the amino acid sequence of an alternateKabat-Chothia Composite CDR-H1 of a humanized 3D6 antibody (derived fromhu3D6VHv3b and hu3D6VHv4b).

SEQ ID NO: 61 sets forth the amino acid sequence of an alternate KabatCDR-H2 of a humanized 3D6 antibody (derived from hu3D6VHv1bA11B6G2).

SEQ ID NO: 62 sets forth the amino acid sequence of an alternate KabatCDR-H2 of a humanized 3D6 antibody (derived from hu3D6VHv1c, hu3D6VHv3b,AND hu3D6VHv4b.

SEQ ID NO: 63 sets forth the amino acid sequence of an alternate KabatCDR-H2 of a humanized 3D6 antibody (derived from hu3D6VHv1d, hu3D6VHv1f,hu3D6VHv3c, and hu3D6VHv4c).

SEQ ID NO: 64 sets forth the amino acid sequence of an alternate KabatCDR-H2 of a humanized 3D6 antibody (derived from hu3D6VHv1e).

SEQ ID NO: 65 sets forth the amino acid sequence of an alternate KabatCDR-H3 of a humanized 3D6 antibody (derived from hu3D6VHv1f).

SEQ ID NO:66 sets forth the amino acid sequence of the heavy chainvariable region of the mouse 6A10 antibody.

SEQ ID NO: 67 sets forth the amino acid sequence of Kabat/Chothiacomposite CDR-H1 of the mouse 6A10 antibody.

SEQ ID NO:68 sets forth the amino acid sequence of Kabat CDR-H2 of themouse 6A10 antibody.

SEQ ID NO: 69 sets forth the amino acid sequence of Kabat CDR-H3 of themouse 6A10 antibody.

SEQ ID NO:70 sets for the amino acid sequence of the VH region of mouseantibody (pdb code 1CR9) used as a structure template for heavy chainhumanization.

SEQ ID NO:71 sets forth the consensus amino acid sequence among theheavy chain variable regions of the selected humanized 3D6 antibodies(VHv1, VHv1b, VHv1bA11, VHv1bA11B6G2, VHv1bA11B6H3, VHv1c, VHv1d, VHv1e,VHv1f, VHv2, VHv3, VHv3b, VHv3c, VHv4, VHv4b, VHv4c, and VHv5) (labeled“Majority’ in FIGS. 4A and 4B).

SEQ ID NO: 72 sets forth the amino acid sequence of the heavy chain of achimeric 3D6 antibody.

SEQ ID NO: 73 sets forth the amino acid sequence of the light chain of achimeric 3D6 antibody.

DEFINITIONS

Monoclonal antibodies or other biological entities are typicallyprovided in isolated form. This means that an antibody or otherbiologically entity is typically at least 50% w/w pure of interferingproteins and other contaminants arising from its production orpurification but does not exclude the possibility that the monoclonalantibody is combined with an excess of pharmaceutically acceptablecarrier(s) or other vehicle intended to facilitate its use. Sometimesmonoclonal antibodies are at least 60%, 70%, 80%, 90%, 95% or 99% w/wpure of interfering proteins and contaminants from production orpurification. Often an isolated monoclonal antibody or other biologicalentity is the predominant macromolecular species remaining after itspurification.

Specific binding of an antibody to its target antigen means an affinityand/or avidity of at least 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, or 10¹² M⁻¹.Specific binding is detectably higher in magnitude and distinguishablefrom non-specific binding occurring to at least one unrelated target.Specific binding can be the result of formation of bonds betweenparticular functional groups or particular spatial fit (e.g., lock andkey type) whereas nonspecific binding is usually the result of van derWaals forces. Specific binding does not however necessarily imply thatan antibody binds one and only one target.

The basic antibody structural unit is a tetramer of subunits. Eachtetramer includes two identical pairs of polypeptide chains, each pairhaving one “light” (about 25 kDa) and one “heavy” chain (about 50-70kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. This variable region is initially expressed linkedto a cleavable signal peptide. The variable region without the signalpeptide is sometimes referred to as a mature variable region. Thus, forexample, a light chain mature variable region means a light chainvariable region without the light chain signal peptide. Thecarboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function.

Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 or more amino acids. See generally,Fundamental Immunology, Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989,Ch. 7 (incorporated by reference in its entirety for all purposes).

An immunoglobulin light or heavy chain variable region (also referred toherein as a “light chain variable domain” (“VL domain”) or “heavy chainvariable domain” (“VH domain”), respectively) consists of a “framework”region interrupted by three “complementarity determining regions” or“CDRs.” The framework regions serve to align the CDRs for specificbinding to an epitope of an antigen. The CDRs include the amino acidresidues of an antibody that are primarily responsible for antigenbinding. From amino-terminus to carboxyl-terminus, both VL and VHdomains comprise the following framework (FR) and CDR regions: FR1,CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDRs 1, 2, and 3 of a VL domain arealso referred to herein, respectively, as CDR-L1, CDR-L2, and CDR-L3;CDRs 1, 2, and 3 of a VH domain are also referred to herein,respectively, as CDR-H1, CDR-H2, and CDR-H3.

The assignment of amino acids to each VL and VH domain is in accordancewith any conventional definition of CDRs. Conventional definitionsinclude, the Kabat definition (Kabat, Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.,1987 and 1991), the Chothia definition (Chothia & Lesk, J. Mol. Biol.196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); acomposite of Chothia Kabat CDR in which CDR-H1 is a composite of Chothiaand Kabat CDRs; the AbM definition used by Oxford Molecular's antibodymodelling software; and, the contact definition of Martin et al(bioinfo.org.uk/abs) (see Table 1). Kabat provides a widely usednumbering convention (Kabat numbering) in which corresponding residuesbetween different heavy chains or between different light chains areassigned the same number. When an antibody is said to comprise CDRs by acertain definition of CDRs (e.g., Kabat) that definition specifies theminimum number of CDR residues present in the antibody (i.e., the KabatCDRs). It does not exclude that other residues falling within anotherconventional CDR definition but outside the specified definition arealso present. For example, an antibody comprising CDRs defined by Kabatincludes among other possibilities, an antibody in which the CDRscontain Kabat CDR residues and no other CDR residues, and an antibody inwhich CDR H1 is a composite Chothia-Kabat CDR H1 and other CDRs containKabat CDR residues and no additional CDR residues based on otherdefinitions.

TABLE 1 Conventional Definitions of CDRs Using Kabat Numbering Compositeof Chothia Loop Kabat Chothia & Kabat AbM Contact L1 L24--L34 L24--L34L24--L34 L24--L34 L30--L36 L2 L50--L56 L50--L56 L50--L56 L50--L56L46--L55 L3 L89--L97 L89--L97 L89--L97 L89--L97 L89--L96 H1 H31--H35BH26--H32 . . . H34* H26--H35B* H26--H35B H30--H35B H2 H50--H65 H52--H56H50--H65 H50--H58 H47--H58 H3 H95--H102 H95--H102 H95--H102 H95--H102H93--H101 *CDR-H1 by Chothia can end at H32, H33, or H34 (depending onthe length of the loop). This is because the Kabat numbering schemeplaces insertions of extra residues at 35A and 35B, whereas Chothianumbering places them at 31A and 31B. If neither H35A nor H35B (Kabatnumbering) is present, the Chothia CDR-H1 loop ends at H32. If only H35Ais present, it ends at H33. If both H35A and H35B are present, it endsat H34.

The term “antibody” includes intact antibodies and binding fragmentsthereof. Typically, fragments compete with the intact antibody fromwhich they were derived for specific binding to the target includingseparate heavy chains, light chains Fab, Fab′, F(ab′)₂, F(ab)c, Dabs,nanobodies, and Fv. Fragments can be produced by recombinant DNAtechniques, or by enzymatic or chemical separation of intactimmunoglobulins. The term “antibody” also includes a bispecific antibodyand/or a humanized antibody. A bispecific or bifunctional antibody is anartificial hybrid antibody having two different heavy/light chain pairsand two different binding sites (see, e.g., Songsivilai and Lachmann,Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol.,148:1547-53 (1992)). In some bispecific antibodies, the two differentheavy/light chain pairs include a humanized 3D6 heavy chain/light chainpair and a heavy chain/light chain pair specific for a different epitopeon tau than that bound by 3D6.

In some bispecific antibodies, one heavy chain/light chain pair is ahumanized 3D6 antibody as further disclosed below and the other heavychain/light chain pair is from an antibody that binds to a receptorexpressed on the blood brain barrier, such as an insulin receptor, aninsulin-like growth factor (IGF) receptor, a leptin receptor, or alipoprotein receptor, or a transferrin receptor (Friden et al., Proc.Natl. Acad. Sci. USA 88:4771-4775, 1991; Friden et al., Science259:373-377, 1993). Such a bispecific antibody can be transferred crossthe blood brain barrier by receptor-mediated transcytosis. Brain uptakeof the bispecific antibody can be further enhanced by engineering thebi-specific antibody to reduce its affinity to the blood brain barrierreceptor. Reduced affinity for the receptor resulted in a broaderdistribution in the brain (see, e.g., Atwal et al., Sci. Trans. Med. 3,84ra43, 2011; Yu et al., Sci. Trans. Med. 3, 84ra44, 2011).

Exemplary bispecific antibodies can also be: (1) a dual-variable-domainantibody (DVD-Ig), where each light chain and heavy chain contains twovariable domains in tandem through a short peptide linkage (Wu et al.,Generation and Characterization of a Dual Variable Domain Immunoglobulin(DVD-Ig™) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg(2010)); (2) a Tandab, which is a fusion of two single chain diabodiesresulting in a tetravalent bispecific antibody that has two bindingsites for each of the target antigens; (3) a flexibody, which is acombination of scFvs with a diabody resulting in a multivalent molecule;(4) a so-called “dock and lock” molecule, based on the “dimerization anddocking domain” in Protein Kinase A, which, when applied to Fabs, canyield a trivalent bispecific binding protein consisting of two identicalFab fragments linked to a different Fab fragment; or (5) a so-calledScorpion molecule, comprising, e.g., two scFvs fused to both termini ofa human Fc-region. Examples of platforms useful for preparing bispecificantibodies include BiTE (Micromet), DART (MacroGenics), Fcab and Mab2(F-star), Fc-engineered IgG1 (Xencor) or DuoBody (based on Fab armexchange, Genmab).

The term “epitope” refers to a site on an antigen to which an antibodybinds. An epitope can be formed from contiguous amino acids ornoncontiguous amino acids juxtaposed by tertiary folding of one or moreproteins. Epitopes formed from contiguous amino acids (also known aslinear epitopes) are typically retained on exposure to denaturingsolvents whereas epitopes formed by tertiary folding (also known asconformational epitopes) are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation. Methods ofdetermining spatial conformation of epitopes include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66,Glenn E. Morris, Ed. (1996).

Antibodies that recognize the same or overlapping epitopes can beidentified in a simple immunoassay showing the ability of one antibodyto compete with the binding of another antibody to a target antigen. Theepitope of an antibody can also be defined X-ray crystallography of theantibody bound to its antigen to identify contact residues.Alternatively, two antibodies have the same epitope if all amino acidmutations in the antigen that reduce or eliminate binding of oneantibody reduce or eliminate binding of the other. Two antibodies haveoverlapping epitopes if some amino acid mutations that reduce oreliminate binding of one antibody reduce or eliminate binding of theother.

Competition between antibodies is determined by an assay in which anantibody under test inhibits specific binding of a reference antibody toa common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495,1990). A test antibody competes with a reference antibody if an excessof a test antibody (e.g., at least 2×, 5×, 10×, 20× or 100×) inhibitsbinding of the reference antibody by at least 50% as measured in acompetitive binding assay. Some test antibodies inhibit binding of thereferences antibody by at least 75%, 90% or 99%. Antibodies identifiedby competition assay (competing antibodies) include antibodies bindingto the same epitope as the reference antibody and antibodies binding toan adjacent epitope sufficiently proximal to the epitope bound by thereference antibody for steric hindrance to occur.

The term “pharmaceutically acceptable” means that the carrier, diluent,excipient, or auxiliary is compatible with the other ingredients of theformulation and not substantially deleterious to the recipient thereof.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

An individual is at increased risk of a disease if the subject has atleast one known risk-factor (e.g., genetic, biochemical, family history,and situational exposure) placing individuals with that risk factor at astatistically significant greater risk of developing the disease thanindividuals without the risk factor.

The term “biological sample” refers to a sample of biological materialwithin or obtainable from a biological source, for example a human ormammalian subject. Such samples can be organs, organelles, tissues,sections of tissues, bodily fluids, peripheral blood, blood plasma,blood serum, cells, molecules such as proteins and peptides, and anyparts or combinations derived therefrom. The term biological sample canalso encompass any material derived by processing the sample. Derivedmaterial can include cells or their progeny. Processing of thebiological sample may involve one or more of filtration, distillation,extraction, concentration, fixation, inactivation of interferingcomponents, and the like.

The term “control sample” refers to a biological sample not known orsuspected to include tau-related disease-affected regions, or at leastnot known or suspect to include diseased regions of a given type.Control samples can be obtained from individuals not afflicted with thetau-related disease. Alternatively, control samples can be obtained frompatients afflicted with the tau-related disease. Such samples can beobtained at the same time as a biological sample thought to comprise thetau-related disease or on a different occasion. A biological sample anda control sample can both be obtained from the same tissue. Preferably,control samples consist essentially or entirely of normal, healthyregions and can be used in comparison to a biological sample thought tocomprise tau-related disease-affected regions. Preferably, the tissue inthe control sample is the same type as the tissue in the biologicalsample. Preferably, the tau-related disease-affected cells thought to bein the biological sample arise from the same cell type (e.g., neurons orglia) as the type of cells in the control sample.

The term “disease” refers to any abnormal condition that impairsphysiological function. The term is used broadly to encompass anydisorder, illness, abnormality, pathology, sickness, condition, orsyndrome in which physiological function is impaired, irrespective ofthe nature of the etiology.

The term “symptom” refers to a subjective evidence of a disease, such asaltered gait, as perceived by the subject. A “sign” refers to objectiveevidence of a disease as observed by a physician.

The term “positive response to treatment” refers to a more favorableresponse in an individual patient or average response in a population ofpatients relative to an average response in a control population notreceiving treatment.

For purposes of classifying amino acids substitutions as conservative ornonconservative, amino acids are grouped as follows: Group I(hydrophobic side chains): met, ala, val, leu, ile; Group II (neutralhydrophilic side chains): cys, ser, thr; Group III (acidic side chains):asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V(residues influencing chain orientation): gly, pro; and Group VI(aromatic side chains): trp, tyr, phe. Conservative substitutionsinvolve substitutions between amino acids in the same class.Non-conservative substitutions constitute exchanging a member of one ofthese classes for a member of another.

Percentage sequence identities are determined with antibody sequencesmaximally aligned by the Kabat numbering convention. After alignment, ifa subject antibody region (e.g., the entire mature variable region of aheavy or light chain) is being compared with the same region of areference antibody, the percentage sequence identity between the subjectand reference antibody regions is the number of positions occupied bythe same amino acid in both the subject and reference antibody regiondivided by the total number of aligned positions of the two regions,with gaps not counted, multiplied by 100 to convert to percentage.

Compositions or methods “comprising” or “including” one or more recitedelements may include other elements not specifically recited. Forexample, a composition that “comprises” or “includes” an antibody maycontain the antibody alone or in combination with other ingredients.

Designation of a range of values includes all integers within ordefining the range, and all subranges defined by integers within therange.

Unless otherwise apparent from the context, the term “about” encompassesinsubstantial variations, such as values within a standard margin oferror of measurement (e.g., SEM) of a stated value.

Statistical significance means p≤10.05.

The singular forms of the articles “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” can include a pluralityof compounds, including mixtures thereof.

DETAILED DESCRIPTION

I. General

The invention provides antibodies that bind to tau. Some antibodiesspecifically bind to an epitope within residues 199-213 or 262-276 ofSEQ ID NO:3 (corresponding to residues 257-271 or 320-334, respectively,of SEQ ID NO:1). Some antibodies bind to tau irrespective ofphosphorylation state. Some antibodies of the invention serve to inhibitor delay tau-associated pathologies and associated symptomaticdeterioration. Although an understanding of mechanism is not requiredfor practice of the invention, a reduction in toxicity may occur as aresult of the antibody inducing phagocytosis of tau, inhibiting tau frominter or intramolecular aggregation, or from binding to other molecules,by stabilizing a non-toxic conformation, by inhibiting intercellular orintracellular transmission of pathogenic tau forms, by blockade of tauphosphorylation, by preventing binding of tau to cells, or by inducingproteolytic cleavage of tau, among other mechanisms. The antibodies ofthe invention or agents that induce such antibodies can be used inmethods of treating or effecting prophylaxis of Alzheimer's and otherdiseases associated with tau.

II. Target Molecules

Unless otherwise apparent from the context, reference to tau means anatural human form of tau including all isoforms irrespective of whetherposttranslational modification (e.g., phosphorylation, glycation, oracetylation) is present. There are six major isoforms (splice variants)of tau occurring in the human brain. The longest of these variants has441 amino acids, of which the initial met residue is cleaved. Residuesare numbered according to the 441 isoform. Thus, for example, referenceto a phosphorylation at position 404 means position 404 of the 441isoform, or corresponding position of any other isoform when maximallyaligned with the 441 isoform. The amino acid sequences of the isoformsand Swiss-Prot numbers are indicated below.

P10636-8 (SEQ ID NO: 1)        10         20         30         40         50         60MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG        70         80         90        100        110        120SETSDAKSTP TAEDVTAPLV DEGAPGKQAĀ AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG       130        140        150        160        170        180HVTQARMVSK SKDGTGSDDK KAKGADGKTK IATPRGAAPP GQKGQANATR IPAKTPPAPK       190        200        210        220        230        240TPPSSGEPPK SGDRSGYSSP GSPGTPGSRS RTPSLPTPPT REPKKVAVVR TPPKSPSSAK       250        260        270        280        290        300SRLQTAPVPM PDLKNVKSKĪ GSTENLKHQP GGGKVQIINK KLDLSNVQSK CGSKDNIKHV       310        320        330        340        350        360PGGGSVQIVY KPVDLSKVTS KCGSLGNIHH KPGGGQVEVK SEKLDFKDRV QSKIGSLDNĪ       370        380        390        400        410        420THVPGGGNKK IETHKLTFRĒ NAKAKTDHGĀ EIVYKSPVVS GDTSPRHLSN VSSTGSIDMV       430        440 DSPQLATLAD EVSASLAKQG L P10636-7 (SEQ ID NO: 2)        10         20         30         40         50         60MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG        70         80         90        100        110        120SETSDAKSTP TAEAEEAGIG DTPSLEDEAĀ GHVTQARMVS KSKDGTGSDD KKAKGADGKT       130        140        150        160        170        180KIATPRGAAP PGQKGQANAT RIPAKTPPAP KTPPSSGEPP KSGDRSGYSS PGSPGTPGSR       190        200        210        220        230        240SRTPSLPTPP TREPKKVAVV RTPPKSPSSĀ KSRLQTAPVP MPDLKNVKSK IGSTENLKHQ       250        260        270        280        290        300PGGGKVQIIN KKLDLSNVQS KCGSKDNIKH VPGGGSVQIV YKPVDLSKVT SKCGSLGNIH       310        320        330        340        350        360HKPGGGQVEV KSEKLDFKDR VQSKIGSLDN ITHVPGGGNK KIETHKLTFR ENAKAKTDHG       370        380        390        400        410AEIVYKSPVV SGDTSPRHLS NVSSTGSIDM VDSPQLATLĀ DEVSASLAKQ GLP10636-6 (4R0N human tau) (SEQ ID NO: 3)        10         20         30         40         50         60MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKAEEAGĪ GDTPSLEDEĀ        70        80          90        100        110        120AGHVTQARMV SKSKDGTGSD DKKAKGADGK TKIATPRGAĀ PPGQKGQANĀ TRIPAKTPPĀ       130        140        150        160        170        180PKTPPSSGEP PKSGDRSGYS SPGSPGTPGS RSRTPSLPTP PTREPKKVAV VRTPPKSPSS       190        200        210        220        230        240AKSRLQTAPV PMPDLKNVKS KIGSTENLKH QPGGGKVQIĪ NKKLDLSNVQ SKCGSKDNIK       250        260        270        280        290        300HVPGGGSVQĪ VYKPVDLSKV TSKCGSLGNĪ HHKPGGGQVĒ VKSEKLDFKD RVQSKIGSLD       310        320        330        340        350        360NITHVPGGGN KKIETHKLTF RENAKAKTDH GAEIVYKSPV VSGDTSPRHL SNVSSTGSID       370        380 MVDSPQLATL ADEVSASLAK QGL P10636-5 (SEQ ID NO: 4)        10         20         30         40         50         60MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG        70         80         90        100        110        120SETSDAKSTP TAEDVTAPLV DEGAPGKQAĀ AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG       130        140        150        160        170        180HVTQARMVSK SKDGTGSDDK KAKGADGKTK IATPRGAAPP GQKGQANATR IPAKTPPAPK       190        200        210        220        230        240TPPSSGEPPK SGDRSGYSSP GSPGTPGSRS RTPSLPTPPT REPKKVAVVR TPPKSPSSAK       250        260        270        280        290        300SRLQTAPVPM PDLKNVKSKĪ GSTENLKHQP GGGKVQIVYK PVDLSKVTSK CGSLGNIHHK       310        320        330        340        350        360PGGGQVEVKS EKLDFKDRVQ SKIGSLDNIT HVPGGGNKKĪ ETHKLTFREN AKAKTDHGAĒ       370        380        390        400        410IVYKSPVVSG DTSPRHLSNV SSTGSIDMVD SPQLATLADĒ VSASLAKQGL P10636-4(SEQ ID NO: 5)        10         20         30         40         50         60MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG        70         80         90        100        110        120SETSDAKSTP TAEAEEAGIG DTPSLEDEAĀ GHVTQARMVS KSKDGTGSDD KKAKGADGKT       130        140        150        160        170        180KIATPRGAAP PGQKGQANAT RIPAKTPPAP KTPPSSGEPP KSGDRSGYSS PGSPGTPGSR       190        200        210        220        230        240SRTPSLPTPP TREPKKVAVV RTPPKSPSSĀ KSRLQTAPVP MPDLKNVKSK IGSTENLKHQ       250        260        270        280        290        300PGGGKVQIVY KPVDLSKVTS KCGSLGNIHH KPGGGQVEVK SEKLDFKDRV QSKIGSLDNĪ       310        320        330        340        350        360THVPGGGNKK IETHKLTFRĒ NAKAKTDHGĀ EIVYKSPVVS GDTSPRHLSN VSSTGSIDMV       370        380 DSPQLATLAD EVSASLAKQG L P10636-2 (SEQ ID NO: 6)10         20         30         40         50         60MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKAEEAGI GDTPSLEDEA        70         80         90        100        110        120AGHVTQARMV SKSKDGTGSD DKKAKGADGK TKIATPRGAA PPGQKGQANA TRIPAKTPPA       130        140        150        160        170        180PKTPPSSGEP PKSGDRSGYS SPGSPGTPGS RSRTPSLPTP PTREPKKVAV VRTPPKSPSS       190        200        210        220        230        240AKSRLQTAPV PMPDLKNVKS KIGSTENLKH QPGGGKVQIV YKPVDLSKVT SKCGSLGNIH       250        260        270        280        290        300HKPGGGQVEV KSEKLDFKDR VQSKIGSLDN ITHVPGGGNK KIETHKLTFR ENAKAKTDHG       310        320        330        340        350AEIVYKSPVV SGDTSPRHLS NVSSTGSIDM VDSPQLATLA DEVSASLAKQ GL

Reference to tau includes known natural variations about 30 of which arelisted in the Swiss-Prot database and permutations thereof, as well asmutations associated with tau pathologies, such as dementia, Pick'sdisease, supranuclear palsy, etc. (see, e.g., Swiss-Prot database andPoorkaj, et al. Ann Neurol. 43:815-825 (1998)). Some examples of taumutations numbered by the 441 isoform are a lysine to threonine mutationat amino acid residue 257 (K257T), an isoleucine to valine mutation atamino acid position 260 (1260V); a glycine to valine mutation at aminoacid position 272 (G272V); an asparagine to lysine mutation at aminoacid position 279 (N279K); an asparagine to histidine mutation at aminoacid position 296 (N296H); a proline to serine mutation at amino acidposition 301 (P301S); a proline to leucine mutation at amino acid 301(P301L); a glycine to valine mutation at amino acid position 303(G303V); a serine to asparagine mutation at position 305 (5305N); aglycine to serine mutation at amino acid position 335 (G335S); a valineto methionine mutation at position 337 (V337M); a glutamic acid tovaline mutation at position 342 (E342V); a lysine to isoleucine mutationat amino acid position 369 (K3691); a glycine to arginine mutation atamino acid position 389 (G389R); and an arginine to tryptophan mutationat amino acid position 406 (R406W).

Tau can be phosphorylated at one or more amino acid residues includingtyrosine at amino acid positions 18, 29, 97, 310, and 394 serine atamino acid positions 184, 185, 198, 199, 202, 208, 214, 235, 237, 238,262, 293, 324, 356, 396, 400, 404, 409, 412, 413, and 422; and threonineat amino acids positions 175, 181, 205, 212, 217, 231, and 403.

Unless otherwise apparent from context, reference to tau, or theirfragments includes the natural human amino acid sequences includingisoforms, mutants, and allelic variants thereof.

III. Antibodies

A. Binding Specificity and Functional Properties

The invention provides antibodies that bind to tau. Some antibodiesspecifically bind to an epitope within residues 199-213 of 383 aminoacid 4R0N human tau protein (SEQ ID NO:3) (corresponding to residues257-271 of SEQ ID NO:1). Some antibodies specifically bind to an epitopewithin residues 262-276 of 383 amino acid 4R0N human tau protein (SEQ IDNO:3) (corresponding to residues 320-334 of SEQ ID NO:1). Someantibodies bind to tau irrespective of phosphorylation state. Someantibodies bind to an epitope not including a residue subject tophosphorylation. These antibodies can be obtained by immunizing with atau polypeptide purified from a natural source or recombinantlyexpressed. Antibodies can be screened for binding tau inunphosphorylated form as well as a form in which one or more residuessusceptible to phosphorylation are phosphorylated. Such antibodiespreferably bind with indistinguishable affinities or at least within afactor of 1.5, 2 or 3-fold to phosphorylated tau compared tonon-phosphorylated tau (i.e., are “pan-specific”). 3D6 is an example ofa pan-specific monoclonal antibody. The invention also providesantibodies binding to the same epitope as any of the foregoingantibodies, such as, for example, the epitope of 3D6. Also included areantibodies competing for binding to tau with any of the foregoingantibodies, such as, for example, competing with 3D6.

The above-mentioned antibodies can be generated de novo by immunizingwith a peptide including residues 199-213 or 262-276 of SEQ ID NO:3(corresponding to residues 257-271 or 320-334, respectively, of SEQ IDNO:1) or by immunizing with a full length tau polypeptide or fragmentthereof comprising such residues and screening for specific binding to apeptide including such residues. Such peptides are preferably attachedto a heterologous conjugate molecule that helps elicit an antibodyresponse to the peptide. Attachment can be direct or via a spacerpeptide or amino acid. Cysteine is used as a spacer amino acid becauseits free SH group facilitates attachment of a carrier molecule. Apolyglycine linker (e.g., 2-6 glycines), with or without a cysteineresidue between the glycines and the peptide can also be used. Thecarrier molecule serves to provide a T-cell epitope that helps elicit anantibody response against the peptide. Several carriers are commonlyused particularly keyhole limpet hemocyanin (KLH), ovalbumin and bovineserum albumin (BSA). Peptide spacers can be added to peptide immunogenas part of solid phase peptide synthesis. Carriers are typically addedby chemical cross-linking. Some examples of chemical crosslinkers thatcan be used include cross-N-maleimido-6-aminocaproyl ester orm-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) (see for example,Harlow, E. et al., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. 1988; Sinigaglia et al.,Nature, 336:778-780 (1988); Chicz et al., J. Exp. Med., 178:27-47(1993); Hammer et al., Cell 74:197-203 (1993); Falk K. et al.,Immunogenetics, 39:230-242 (1994); WO 98/23635; and, Southwood et al. J.Immunology, 160:3363-3373 (1998)). The carrier and spacer if present canbe attached to either end of the immunogen.

A peptide with optional spacer and carrier can be used to immunizelaboratory animals or B-cells as described in more detail below.Hybridoma supernatants can be tested for ability to bind one or morepeptides including residues 199-213 or 262-276 of SEQ ID NO:3(corresponding to residues 257-271 or 320-334, respectively, of SEQ IDNO:1) and/or phosphorylated and non-phosphorylated forms of tau, suchas, for example, a full-length isoform of tau with position 404 inphosphorylated form. The peptide can be attached to a carrier or othertag to facilitate the screening assay. In this case, the carrier or tagis preferentially different than the combination of spacer and carriermolecule used for immunization to eliminate antibodies specific for thespacer or carrier rather than the tau peptide. Any of the tau isoformscan be used.

The invention provides monoclonal antibodies binding to epitopes withintau. An antibody designated 3D6 is one such exemplary mouse antibody.Unless otherwise apparent from context, reference to 3D6 should beunderstood as referring to any of the mouse, chimeric, veneered, andhumanized forms of this antibody. The antibody has been deposited as[DEPOSIT NUMBER]. This antibody specifically binds within amino acidresidues 199-213 or 262-276 of the 383 amino acid 4R0N human tau protein(SEQ ID NO:3) (corresponding to amino acid residues 257-271 or 320-334,respectively, of SEQ ID NO:1). This antibody is further characterized byits ability to bind both phosphorylated and unphosphorylated tau, bothnon-pathological and pathological forms and conformations of tau, andmisfolded/aggregated forms of tau. An antibody designated 6A10 is onesuch exemplary mouse antibody. Unless otherwise apparent from context,reference to 6A10 should be understood as referring to any of the mouse,chimeric, veneered, and humanized forms of this antibody. Kabat/ChothiaComposite CDRs of the heavy chain of 6A10 are designated SEQ ID NOs: 67,68, and 69, respectively, and Kabat CDRs of the light chain of 6A10 aredesignated SEQ ID NOs: 12, 13, and 14, respectively. Mouse 6A10 shares82.1% of VH sequence identity and 100% VL sequence identity with the VHchain and VL chain, respectively, of mouse 3D6.

Some antibodies of the invention bind to the same or overlapping epitopeas an antibody designated 3D6. The sequences of the heavy and lightchain mature variable regions of this antibody are designated SEQ IDNOs: 7 and 11, respectively. Other antibodies having such a bindingspecificity can be produced by immunizing mice with tau or a portionthereof including the desired epitope (e.g. 199-213 or 262-276 of SEQ IDNO:3, corresponding to residues 257-271 or 320-334, respectively, of SEQID NO:1) and screening resulting antibodies for binding to tauoptionally in competition with an antibody having the variable regionsof mouse 3D6 (IgG1 kappa). Fragments of tau including the desiredepitope can be linked to a carrier that helps elicit an antibodyresponse to the fragment and/or be combined with an adjuvant the helpselicit such a response. Such antibodies can be screened for differentialbinding to tau or a fragment thereof compared with mutants of specifiedresidues. Screening against such mutants more precisely defines thebinding specificity to allow identification of antibodies whose bindingis inhibited by mutagenesis of particular residues and which are likelyto share the functional properties of other exemplified antibodies. Themutations can be systematic replacement substitution with alanine (orserine if an alanine is present already) one residue at a time, or morebroadly spaced intervals, throughout the target or throughout a sectionthereof in which an epitope is known to reside. If the same set ofmutations significantly reduces the binding of two antibodies, the twoantibodies bind the same epitope.

Antibodies having the binding specificity of a selected murine antibody(e.g., 3D6) can also be produced using a variant of the phage displaymethod. See Winter, WO 92/20791. This method is particularly suitablefor producing human antibodies. In this method, either the heavy orlight chain variable region of the selected murine antibody is used as astarting material. If, for example, a light chain variable region isselected as the starting material, a phage library is constructed inwhich members display the same light chain variable region (i.e., themurine starting material) and a different heavy chain variable region.The heavy chain variable regions can for example be obtained from alibrary of rearranged human heavy chain variable regions. A phageshowing strong specific binding for tau or a fragment thereof (e.g., atleast 10⁸ and preferably at least 10⁹ M⁻¹) is selected. The heavy chainvariable region from this phage then serves as a starting material forconstructing a further phage library. In this library, each phagedisplays the same heavy chain variable region (i.e., the regionidentified from the first display library) and a different light chainvariable region. The light chain variable regions can be obtained forexample from a library of rearranged human variable light chain regions.Again, phage showing strong specific binding for tau or a fragmentthereof are selected. The resulting antibodies usually have the same orsimilar epitope specificity as the murine starting material.

Kabat/Chothia Composite CDRs of the heavy chain of 3D6 are designatedSEQ ID NOs: 8, 9, and 10, respectively, and Kabat CDRs of the lightchain of 3D6 are designated SEQ ID NOs: 12, 13, and 14, respectively.

Table 2 indicates the 3D6 CDRs as defined by Kabat, Chothia, Compositeof Chothia and Kabat (also referred to herein as “Kabat/ChothiaComposite”), AbM, and Contact.

TABLE 2 3D6 CDRs as defined by Kabat, Chothia, Composite of Chothia andKabat, AbM, and Contact Composite of Chothia Loop Kabat Chothia & KabatAbM Contact L1 L24--L34 L24--L34 L24--L34 L24--L34 L30--L36 SEQ ID NO:12 SEQ ID NO: 12 SEQ ID NO: 12 SEQ ID NO: 12 SEQ ID NO: 36 L2 L50--L56L50--L56 L50--L56 L50--L56 L46--L55 SEQ ID NO: 13 SEQ ID NO: 13 SEQ IDNO: 13 SEQ ID NO: 13 SEQ ID NO: 37 L3 L89--L97 L89--L97 L89--L97L89--L97 L89--L96 SEQ ID NO: 14 SEQ ID NO: 14 SEQ ID NO: 14 SEQ ID NO:14 SEQ ID NO: 38 H1 H31--H35B H26--H32 H26--H35B H26-H35B H30--H35B SEQID NO: 32 SEQ ID NO: 33 SEQ ID NO: 8 SEQ ID NO: 8 SEQ ID NO: 39 H2H50--H65 H52--H56 H50--H65 H50--H58 H47--H58 SEQ ID NO: 9 SEQ ID NO: 34SEQ ID NO: 9 SEQ ID NO: 35 SEQ ID NO: 40 H3 H95--H102 H95--H102H95--H102 H95--H102 H93--H101 SEQ ID NO: 10 SEQ ID NO: 10 SEQ ID NO: 10SEQ ID NO: 10 SEQ ID NO: 41

Other antibodies can be obtained by mutagenesis of cDNA encoding theheavy and light chains of an exemplary antibody, such as 3D6. Monoclonalantibodies that are at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%identical to 3D6 in amino acid sequence of the mature heavy and/or lightchain variable regions and maintain its functional properties, and/orwhich differ from the respective antibody by a small number offunctionally inconsequential amino acid substitutions (e.g.,conservative substitutions), deletions, or insertions are also includedin the invention. Monoclonal antibodies having at least one or all sixCDR(s) as defined by any conventional definition, but preferably Kabat,that are 90%, 95%, 99% or 100% identical to corresponding CDRs of 3D6are also included.

The invention also provides antibodies having some or all (e.g., 3, 4,5, and 6) CDRs entirely or substantially from 3D6. Such antibodies caninclude a heavy chain variable region that has at least two, and usuallyall three, CDRs entirely or substantially from the heavy chain variableregion of 3D6 and/or a light chain variable region having at least two,and usually all three, CDRs entirely or substantially from the lightchain variable region of 3D6. The antibodies can include both heavy andlight chains. A CDR is substantially from a corresponding 3D6 CDR whenit contains no more than 4, 3, 2, or 1 substitutions, insertions, ordeletions, except that CDR-H2 (when defined by Kabat) can have no morethan 6, 5, 4, 3, 2, or 1 substitutions, insertions, or deletions. Suchantibodies can have at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%identity to 3D6 in the amino acid sequence of the mature heavy and/orlight chain variable regions and maintain their functional properties,and/or differ from 3D6 by a small number of functionally inconsequentialamino acid substitutions (e.g., conservative substitutions), deletions,or insertions.

Some antibodies identified by such assays can bind to monomeric,misfolded, aggregated, phosphorylated, or unphosphorylated forms of tauor otherwise. Likewise, some antibodies are immunoreactive onnon-pathological and pathological forms and conformations of tau.

B. Non-Human Antibodies

The production of other non-human antibodies, e.g., murine, guinea pig,primate, rabbit or rat, against tau or a fragment thereof (e.g., aminoacid residues 199-213 or 262-276 of SEQ ID NO:3) (corresponding to aminoacid residues 257-271 or 320-334, respectively, of SEQ ID NO:1) can beaccomplished by, for example, immunizing the animal with tau or afragment thereof. See Harlow & Lane, Antibodies, A Laboratory Manual(CSHP NY, 1988) (incorporated by reference for all purposes). Such animmunogen can be obtained from a natural source, by peptide synthesis,or by recombinant expression. Optionally, the immunogen can beadministered fused or otherwise complexed with a carrier protein.Optionally, the immunogen can be administered with an adjuvant. Severaltypes of adjuvant can be used as described below. Complete Freund'sadjuvant followed by incomplete adjuvant is preferred for immunizationof laboratory animals. Rabbits or guinea pigs are typically used formaking polyclonal antibodies. Mice are typically used for makingmonoclonal antibodies. Antibodies are screened for specific binding totau or an epitope within tau (e.g., an epitope comprising one or more ofamino acid residues 199-213 or 262-276 of SEQ ID NO:3) (corresponding toamino acid residues 257-271 or 320-334, respectively, of SEQ ID NO:1).Such screening can be accomplished by determining binding of an antibodyto a collection of tau variants, such as tau variants containing aminoacid residues 199-213 or 262-276 of SEQ ID NO: 3 (corresponding to aminoacid residues 257-271 or 320-334, respectively, of SEQ ID NO:1) ormutations within these residues, and determining which tau variants bindto the antibody. Binding can be assessed, for example, by Western blot,FACS or ELISA.

C. Humanized Antibodies

A humanized antibody is a genetically engineered antibody in which CDRsfrom a non-human “donor” antibody are grafted into human “acceptor”antibody sequences (see, e.g., Queen, U.S. Pat. Nos. 5,530,101 and5,585,089; Winter, U.S. Pat. No. 5,225,539; Carter, U.S. Pat. No.6,407,213; Adair, U.S. Pat. No. 5,859,205; and Foote, U.S. Pat. No.6,881,557). The acceptor antibody sequences can be, for example, amature human antibody sequence, a composite of such sequences, aconsensus sequence of human antibody sequences, or a germline regionsequence. Thus, a humanized antibody is an antibody having at leastthree, four, five or all CDRs entirely or substantially from a donorantibody and variable region framework sequences and constant regions,if present, entirely or substantially from human antibody sequences.Similarly a humanized heavy chain has at least one, two and usually allthree CDRs entirely or substantially from a donor antibody heavy chain,and a heavy chain variable region framework sequence and heavy chainconstant region, if present, substantially from human heavy chainvariable region framework and constant region sequences. Similarly ahumanized light chain has at least one, two and usually all three CDRsentirely or substantially from a donor antibody light chain, and a lightchain variable region framework sequence and light chain constantregion, if present, substantially from human light chain variable regionframework and constant region sequences. Other than nanobodies and dAbs,a humanized antibody comprises a humanized heavy chain and a humanizedlight chain. A CDR in a humanized antibody is substantially from acorresponding CDR in a non-human antibody when at least 85%, 90%, 95% or100% of corresponding residues (as defined by any conventionaldefinition but preferably defined by Kabat) are identical between therespective CDRs. The variable region framework sequences of an antibodychain or the constant region of an antibody chain are substantially froma human variable region framework sequence or human constant regionrespectively when at least 85%, 90%, 95% or 100% of correspondingresidues defined by Kabat are identical. To be classified as humanizedunder the 2014 World Health Organization (WHO) Internationalnon-proprietary names (INN) definition of humanized antibodies, anantibody must have at least 85% identity to human germline antibodysequences (i.e., prior to somatic hypermutation). Mixed antibodies areantibodies for which one antibody chain (e.g., heavy chain) meets thethreshold but the other chain (e.g., light chain) does not meet thethreshold. An antibody is classified as chimeric if neither chain meetsthe threshold, even though the variable framework regions for bothchains were substantially human with some murine backmutations. See,Jones et al. (2016) The INNs and outs of antibody nonproprietary names,mAbs 8:1, 1-9, DOI: 10.1080/19420862.2015.1114320. See also “WHO-INN:International nonproprietary names (INN) for biological andbiotechnological substances (a review)” (Internet) 2014. Available from:World Health Organization website), incorporated herein by reference.For the avoidance of doubt, the term “humanized” as used herein is notintended to be limited to the 2014 WHO INN definition of humanizedantibodies. Some of the humanized antibodies provided herein have atleast 85% sequence identity to human germline sequences and some of thehumanized antibodies provided herein have less than 85% sequenceidentity to human germline sequences. Some of the heavy chains of thehumanized antibodies provided herein have from about 60% to 100%sequence identity to human germ line sequences, such as, for example, inthe range of about 60% to 69%, 70% to 79%, 80% to 84%, or 85% to 89%.Some heavy chains fall below the 2014 WHO INN definition and have, forexample, about 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, or 82%, 83%, or 84% sequence identityto human germ line sequences, while other heavy chains meet the 2014 WHOINN definition and have about 85%, 86%, 87%, 88%, 89% or greatersequence identity to human germ line sequences. Some of the light chainsof the humanized antibodies provided herein have from about 60% to 100%sequence identity to human germ line sequences, such as, for example, inthe range of about 80% to 84% or 85% to 89%. Some light chains fallbelow the 2014 WHO INN definition and have, for example, about 81%, 82%,83% or 84% sequence identity to human germ line sequences, while otherlight chains meet the 2014 WHO INN definition and have about 85%, 86%,87%, 88%, 89% or greater sequence identity to human germ line sequences.Some humanized antibodies provided herein that are “chimeric” under the2014 WHO INN definition have heavy chains with less than 85% identity tohuman germ line sequences paired with light chains having less than 85%identity to human germ line sequences. Some humanized antibodiesprovided herein are “mixed” under the 2014 WHO INN definition, forexample, having a heavy chain with at least 85% sequence identity tohuman germ line sequences paired with a light chain having less than 85%sequence identity to human germ line sequences, or vice versa. Somehumanized antibodies provided herein meet the 2014 WHO INN definition of“humanized” and have a heavy chain with at least 85% sequence identityto human germ line sequences paired with a light chain having at least85% sequence identity to human germ line sequences. Additional humanizedantibodies of the invention meet the 2014 WHO INN definition of “mixed”and include antibodies having a mature heavy chain having an amino acidsequence of any of SEQ ID NOs:15-19 and SEQ ID NOs:46-57 paired with amature light chain having an amino acid sequence of any of SEQ IDNOs:20-23.

Although humanized antibodies often incorporate all six CDRs (defined byany conventional definition but preferably as defined by Kabat) from amouse antibody, they can also be made with less than all CDRs (e.g., atleast 3, 4, or 5 CDRs) from a mouse antibody (e.g., Pascalis et al., J.Immunol. 169:3076, 2002; Vajdos et al., J. of Mol. Biol., 320: 415-428,2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; Tamura et al,J. Immunol., 164:1432-1441, 2000).

In some antibodies only part of the CDRs, namely the subset of CDRresidues required for binding, termed the SDRs, are needed to retainbinding in a humanized antibody. CDR residues not contacting antigen andnot in the SDRs can be identified based on previous studies (for exampleresidues H60-H65 in CDR H2 are often not required), from regions ofKabat CDRs lying outside Chothia hypervariable loops (Chothia, J. Mol.Biol. 196:901, 1987), by molecular modeling and/or empirically, or asdescribed in Gonzales et al., Mol. Immunol. 41: 863, 2004. In suchhumanized antibodies at positions in which one or more donor CDRresidues is absent or in which an entire donor CDR is omitted, the aminoacid occupying the position can be an amino acid occupying thecorresponding position (by Kabat numbering) in the acceptor antibodysequence. The number of such substitutions of acceptor for donor aminoacids in the CDRs to include reflects a balance of competingconsiderations. Such substitutions are potentially advantageous indecreasing the number of mouse amino acids in a humanized antibody andconsequently decreasing potential immunogenicity and/or for meeting theWHO INN definition of “humanized”. However, substitutions can also causechanges of affinity, and significant reductions in affinity arepreferably avoided. Positions for substitution within CDRs and aminoacids to substitute can also be selected empirically.

The human acceptor antibody sequences can optionally be selected fromamong the many known human antibody sequences to provide a high degreeof sequence identity (e.g., 65-85% identity) between a human acceptorsequence variable region frameworks and corresponding variable regionframeworks of a donor antibody chain.

An example of an acceptor sequence for the heavy chain is the humanmature heavy chain variable region with NCBI accession code BAC01986.1(SEQ ID NO:24). An example of an acceptor sequence for the heavy chainis the human mature heavy chain variable region with NCBI accession codeIMGT #IGHV1-69-2*01 (SEQ ID NO:25). An example of an acceptor sequencefor the heavy chain is the human mature heavy chain variable region withNCBI accession code IMGT #IGKJ1*01 (SEQ ID NO:26). IMGT #IGHV1-69-2*01(SEQ ID NO:25) shares the canonical form of mouse 3D6 heavy chain CDR-H1and H2. IMGT #IGHV1-69-2*01 (SEQ ID NO:25) and IMGT #IGKJ1*01 (SEQ IDNO:26) and both belong to human heavy chain subgroup 1. An example of anacceptor sequence for the light chain is the human mature light chainvariable region with NCBI accession code IMGT #IGKV2-30*02 (SEQ IDNO:27). An example of an acceptor sequence for the light chain is thehuman mature light chain variable region with NCBI accession code IMGT#IGKJ2*01 (SEQ ID NO:28). An example of an acceptor sequence for thelight chain is the human mature light chain variable region with NCBIaccession code AAZ09048.1 (SEQ ID NO:29). IMGT #IGKV2-30*02 (SEQ IDNO:27) has the same canonical classes for CDR-L1, CDR-L2 and L3 as mouse3D6. IMGT #IGKV2-30*02 (SEQ ID NO:27) and IMGT #IGKJ2*01 (SEQ ID NO:28)belong to human kappa subgroup 2.

If more than one human acceptor antibody sequence is selected, acomposite or hybrid of those acceptors can be used, and the amino acidsused at different positions in the humanized light chain and heavy chainvariable regions can be taken from any of the human acceptor antibodysequences used. For example, the human mature heavy chain variableregions with NCBI accession codes IMGT #IGHV1-69-2*01 (SEQ ID NO:25) andBAC01986.1 (SEQ ID NO:24) were used as acceptor sequences forhumanization of the 3D6 mature heavy chain variable region. Examples ofpositions in which these two acceptors differ include positions H17 (Tor S) and H20 (I or V). Humanized versions of the 3D6 heavy chainvariable region can include either amino acid at either of thesepositions.

Certain amino acids from the human variable region framework residuescan be selected for substitution based on their possible influence onCDR conformation and/or binding to antigen. Investigation of suchpossible influences is by modeling, examination of the characteristicsof the amino acids at particular locations, or empirical observation ofthe effects of substitution or mutagenesis of particular amino acids.

For example, when an amino acid differs between a murine variable regionframework residue and a selected human variable region frameworkresidue, the human framework amino acid can be substituted by theequivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid:

-   -   (1) noncovalently binds antigen directly;    -   (2) is adjacent to a CDR region or within a CDR as defined by        Chothia but not Kabat;    -   (3) otherwise interacts with a CDR region (e.g., is within about        6 Å of a CDR region), (e.g., identified by modeling the light or        heavy chain on the solved structure of a homologous known        immunoglobulin chain); or    -   (4) is a residue participating in the VL-VH interface.

The invention provides humanized forms of the murine 3D6 antibodyincluding 17 exemplified humanized heavy chain mature variable regions(hu3D6VHv1 (SEQ ID NO:15), hu3D6VHv2 (SEQ ID NO:16), hu3D6VHv1b (SEQ IDNO:17), hu3D6VHv1bA11 (SEQ ID NO:18), hu3D6VHv5 (SEQ ID NO:19),hu3D6VHv1bA11B6G2 (SEQ ID NO:46), hu3D6VHv1bA11B6H3 (SEQ ID NO:47),hu3D6VHv1c (SEQ ID NO:48), hu3D6VHv1d (SEQ ID NO:49), hu3D6VHv1e (SEQ IDNO:50), hu3D6VHv1f (SEQ ID NO:51), hu3D6VHv3 (SEQ ID NO:52), hu3D6VHv3b(SEQ ID NO:53), hu3D6VHv3c (SEQ ID NO:54), hu3D6VHv4 (SEQ ID NO:55),hu3D6VHv4b (SEQ ID NO:56), and hu3D6VHv4c (SEQ ID NO:57)) and 4exemplified humanized light chain mature variable regions (hu3D6VLv1(SEQ ID NO:20), hu3D6VLv2 (SEQ ID NO:21, hu3D6VLv3 (SEQ ID NO:22), andhu3D6VLv4 (SEQ ID NO:23)).

In an embodiment, humanized sequences are generated using a two-stagePCR protocol that allows introduction of multiple mutations, deletions,and insertions using QuikChange site-directed mutagenesis [Wang, W. andMalcolm, B. A. (1999) BioTechniques 26:680-682)].

Framework residues from classes (1) through (3) as defined by Queen,U.S. Pat. No. 5,530,101, are sometimes alternately referred to ascanonical and vernier residues. Framework residues that help define theconformation of a CDR loop are sometimes referred to as canonicalresidues (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Thornton &Martin, J. Mol. Biol. 263:800-815 (1996)). Framework residues thatsupport antigen-binding loop conformations and play a role infine-tuning the fit of an antibody to antigen are sometimes referred toas vernier residues (Foote & Winter, J. Mol. Biol 224:487-499 (1992)).

Other framework residues that are candidates for substitution areresidues creating a potential glycosylation site. Still other candidatesfor substitution are acceptor human framework amino acids that areunusual for a human immunoglobulin at that position. These amino acidscan be substituted with amino acids from the equivalent position of themouse donor antibody or from the equivalent positions of more typicalhuman immunoglobulins.

Other framework residues that are candidates for substitution areN-terminal glutamine residues (Q) that may be replaced with glutamicacid (E) to minimize potential for pyroglutamate conversion [Y. DianaLiu, et al., 2011, J. Biol. Chem., 286: 11211-11217]. Glutamic acid (E)conversion to pyroglutamate (pE) occurs more slowly than from glutamine(Q). Because of the loss of a primary amine in the glutamine to pEconversion, antibodies become more acidic. Incomplete conversionproduces heterogeneity in the antibody that can be observed as multiplepeaks using charge-based analytical methods. Heterogeneity differencesmay indicate a lack of process control.

Exemplary humanized antibodies are humanized forms of the mouse 3D6,designated Hu3D6.

The mouse antibody 3D6 comprises mature heavy and light chain variableregions having amino acid sequences comprising SEQ ID NO: 7 and SEQ IDNO:11, respectively. The invention provides 17 exemplified humanizedmature heavy chain variable regions: hu3D6VHv1, hu3D6VHv2, hu3D6VHv1b,hu3D6VHv1bA11, hu3D6VHv5, hu3D6VHv1bA11B6G2, hu3D6VHv1bA11B6H3,hu3D6VHv1c, hu3D6VHv1d, hu3D6VHv1e, hu3D6VHv1f, hu3D6VHv3, hu3D6VHv3b,hu3D6VHv3c, hu3D6VHv4, hu3D6VHv4b, and hu3D6VHv4c. The invention furtherprovides 4 exemplified human mature light chain variable regions:hu3D6VLv1, hu3D6VLv2, hu3D6VLv3, and VLv4. FIGS. 2 and 3 show alignmentsof the heavy chain variable region and light chain variable region,respectively, of murine 3D6 and various humanized antibodies.

For reasons such as possible influence on CDR conformation and/orbinding to antigen, mediating interaction between heavy and lightchains, interaction with the constant region, being a site for desiredor undesired post-translational modification, being an unusual residuefor its position in a human variable region sequence and thereforepotentially immunogenic, getting aggregation potential, and otherreasons, the following 30 variable region framework positions wereconsidered as candidates for substitutions in the 4 exemplified humanmature light chain variable regions and the 17 exemplified human matureheavy chain variable regions, as further specified in the examples: L2(V2I), L7 (S7T), L12 (P12S), L15 (L15I), L36 (F36L), L37 (Q37L), L45(R45K), L60 (D60S), L100 (Q100G), H10 (E10D), H12 (K12V), H13 (K13R),H17 (T17L), H24 (V24A), H38 (Q38R), H40 (A40R), H42 (G42E), H43 (K43Q),H48 (M48I), H66 (R66K), H67 (V67A), H76 (D76N), H80 (M80L), H81 (E81Q),H82a (S82a-G), H83 (R83T), H91 (Y91F), H93 (A93S), H108 (L108T), andH109 (V109L). The following 9 variable region CDR positions wereconsidered as candidates for substitutions in the 17 exemplified humanmature heavy chain variable regions, as further specified in theexamples: H27 (F27Y), H28 (N28T), H29 (I29F), H30 (K30T), H51 (I51V),H54 (N54D), H60 (D60A), H61 (P61E), and H102 (F102Y). In some humanized3D6 antibodies, Kabat-Chothia Composite CDR-H1 has an amino acidsequence comprising SEQ ID NO: 42, and Kabat CDR-H2 has an amino acidsequence comprising SEQ ID NO: 43. In some humanized 3D6 antibodies,Kabat-Chothia Composite CDR-H1 has an amino acid sequence comprising SEQID NO: 42, and Kabat CDR-H2 has an amino acid sequence comprising SEQ IDNO: 61. In some humanized 3D6 antibodies, Kabat-Chothia Composite CDR-H1has an amino acid sequence comprising SEQ ID NO: 58 and Kabat CDR-H2 hasan amino acid sequence comprising SEQ ID NO: 62. In some humanized 3D6antibodies, Kabat-Chothia Composite CDR-H1 has an amino acid sequencecomprising SEQ ID NO: 42, Kabat CDR-H2 has an amino acid sequencecomprising SEQ ID NO: 64, and Kabat CDR-H3 has an amino acid sequencecomprising SEQ ID NO:65. In some humanized 3D6 antibodies, Kabat-ChothiaComposite CDR-H1 has an amino acid sequence comprising SEQ ID NO: 42 andKabat CDR-H2 has an amino acid sequence comprising SEQ ID NO:64. In somehumanized 3D6 antibodies, Kabat-Chothia Composite CDR-H1 has an aminoacid sequence comprising SEQ ID NO: 59 and Kabat CDR-H2 has an aminoacid sequence comprising SEQ ID NO:63. In some humanized 3D6 antibodies,Kabat-Chothia Composite CDR-H1 has an amino acid sequence comprising SEQID NO: 60 and Kabat CDR-H2 has an amino acid sequence comprising SEQ IDNO:62.

Here, as elsewhere, the first-mentioned residue is the residue of ahumanized antibody formed by grafting Kabat CDRs or a compositeChothia-Kabat CDR in the case of CDR-H1 into a human acceptor framework,and the second-mentioned residue is a residue being considered forreplacing such residue. Thus, within variable region frameworks, thefirst mentioned residue is human, and within CDRs, the first mentionedresidue is mouse.

Exemplified antibodies include any permutations or combinations of theexemplified mature heavy and light chain variable regions VHv1/VLv1,VHv1/VLv2, VHv1/VLv3, VHv1/VLv4, VHv2/VLv1, VHv2/VLv2, VHv2/VLv3,VHv2/VLv4, VHv1b/VLv1, VHv1b/VLv2, VHv1b/VLv3, VHv1b/VLv4,VHv1bA11/VLv1, VHv1bA11/VLv2, VHv1bA11/VLv3, VHv1bA11/VLv4, VHv5/VLv1,VHv5/VLv2, VHv5/VLv3, VHv5/VLv4, VHv1bA11B6G2/VLv1, VHv1bA11B6G2/VLv2,VHv1bA11B6G2/VLv3, VHv1bA11B6G2/VLv4, VHv1bA11B6H3/VLv1,VHv1bA11B6H3/VLv2, VHv1bA11B6H3/VLv3, VHv1bA11B6H3/VLv4, VHv1c/VLv1,VHv1c/VLv2, VHv1c/VLv3, VHv1c/VLv4, VHv1d/VLv1, VHv1d/VLv2, VHv1d/VLv3,VHv1d/VLv4, VHv1e/VLv1, VHv1e/VLv2, VHv1e/VLv3, VHv1e/VLv4, VHv1f/VLv1,VHv1f/VLv2, VHv1f/VLv3, VHv1f/VLv4, VHv3/VLv1, VHv3/VLv2, VHv3/VLv3,VHv3/VLv4, VHv3b/VLv1, VHv3b/VLv2, VHv3b/VLv3, VHv3b/VLv4, VHv3c/VLv1,VHv3c/VLv2, VHv3c/VLv3, VHv3c/VLv4, VHv4/VLv1, VHv4/VLv2, VHv4/VLv3,VHv4/VLv4, VHv4b/VLv1, VHv4b/VLv2, VHv4b/VLv3, VHv4b/VLv4, VHv4c/VLv1,VHv4c/VLv2, VHv4c/VLv3, or VHv4c/VLv4.

The invention provides variants of the 3D6 humanized antibody in whichthe humanized mature heavy chain variable region shows at least 90%,95%, 96%, 97%, 98%, or 99% identity to hu3D6VHv1 (SEQ ID NO:15),hu3D6VHv2 (SEQ ID NO:16), hu3D6VHv1b (SEQ ID NO:17), hu3D6VHv1bA11 (SEQID NO:18), hu3D6VHv5 (SEQ ID NO:19), hu3D6VHv1bA11B6G2 (SEQ ID NO:46),hu3D6VHv1bA11B6H3 (SEQ ID NO:47), hu3D6VHv1c (SEQ ID NO:48), hu3D6VHv1d(SEQ ID NO:49), hu3D6VHv1e (SEQ ID NO:50), hu3D6VHv1f (SEQ ID NO:51),hu3D6VHv3 (SEQ ID NO:52), hu3D6VHv3b (SEQ ID NO:53), hu3D6VHv3c (SEQ IDNO:54), hu3D6VHv4 (SEQ ID NO:55), hu3D6VHv4b (SEQ ID NO:56), andhu3D6VHv4c (SEQ ID NO:57) and the humanized mature light chain variableregion shows at least 90%, 95%, 96%, 97%, 98%, or 99% identity tohu3D6VLv1 (SEQ ID NO:20), hu3D6VLv2 (SEQ ID NO:21, hu3D6VLv3 (SEQ IDNO:22), and hu3D6VLv4 (SEQ ID NO:23). In some such antibodies at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,or all 39 of the backmutations or other mutations in SEQ ID NOs:15-19,SEQ ID NO:20-23, and SEQ ID NO:46-57) are retained.

In some humanized 3D6 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:H38 is occupied by R and H93 is occupied by S. In some humanized 3D6antibodies, positions H38 and H93 in the VH region are occupied by, Rand S, respectively.

In some humanized 3D6 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:H38 is occupied by R, H43 is occupied by Q, H83 is occupied by T, andH93 is occupied by S. In some humanized 3D6 antibodies, positions H38,H43, H83, and H93 in the VH region are occupied by, R, Q, T, and S,respectively.

In some humanized 3D6 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:H12 is occupied by V, H24 is occupied by A, H48 is occupied by I, H67 isoccupied by A, H80 is occupied by L, H81 is occupied by Q, and H91 isoccupied by F. In some humanized 3D6 antibodies, positions H12, H24,H48, H67, H80, H81, and H91 in the VH region are occupied by V, A, I, A,L, Q, and F, respectively.

In some humanized 3D6 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:H13 is occupied by R and H66 is occupied by K. In some humanized 3D6antibodies, positions H13 and H66 in the VH region are occupied by R andK, respectively.

In some humanized 3D6 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:H40 is occupied by R and H82a is occupied by G. In some humanized 3D6antibodies, positions H40 and H82a in the VH region are occupied by Rand G, respectively.

In some humanized 3D6 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:H42 is occupied by E and H76 is occupied by N. In some humanized 3D6antibodies, positions H42 and H76 in the VH region are occupied by E andN, respectively.

In some humanized 3D6 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:H40 is occupied by R, H82a is occupied by G, and H83 is occupied by T.In some humanized 3D6 antibodies, positions H40, H82a, and H83 in the VHregion are occupied by R, G, and T, respectively.

In some humanized 3D6 antibodies, H12 in the VH region is occupied by V.

In some humanized 3D6 antibodies, H80 in the VH region is occupied by L.

In some humanized 3D6 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:H24 is occupied by A, H48 is occupied by I, H67 is occupied by A, H80 isoccupied by L, and H91 is occupied by F In some humanized 3D6antibodies, positions H24, H48, H67, H80, and H91 in the VH region areoccupied by A, I, A, L, and F, respectively.

In some humanized 3D6 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:H43 is occupied by Q, and H81 is occupied by Q. In some humanized 3D6antibodies, positions H43 and H81 in the VH region are occupied by Q,and Q, respectively.

In some humanized 3D6 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:H24 is occupied by A, and H91 is occupied by F. In some humanized 3D6antibodies, positions H24 and H91 in the VH region are occupied by A andF, respectively.

In some humanized 3D6 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:H13 is occupied by R, H17 is occupied by L, H29 is occupied by F, H42 isoccupied by E, H43 is occupied by Q, H61 is occupied by E, H76 isoccupied by N, H80 is occupied by L, H81 is occupied by Q. In somehumanized 3D6 antibodies, positions H13, H17, H29, H42, H43, H61, H76,H80, and H81 in the VH region are occupied by R, L, F, E, Q, E, N, L,and Q, respectively.

In some humanized 3D6 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:H24 is occupied by A, H28 is occupied by T, H48 is occupied by I, H54 isoccupied by D, H60 is occupied by A, H67 is occupied by A, H80 isoccupied by L, and H91 is occupied by F. In some humanized 3D6antibodies, positions H24, H28, H48, H54, H60, H67, H80, and H91 in theVH region are occupied by A, T, I, D, A, A, L, and F, respectively.

In some humanized 3D6 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:H10 is occupied by D, H17 is occupied by L, H24 is occupied by A, H28 isoccupied by T, H43 is occupied by Q, H48 is occupied by I, H60 isoccupied by A, H61 is occupied by E, H91 is occupied by F, H108 isoccupied by T, and H109 is occupied by L. In some humanized 3D6antibodies, positions H10, H17, H24, H28, H43, H48, H60, H61, H91, H108,and H109 in the VH region are occupied by D, L, A, T, Q, I, A, E, F, T,and L, respectively.

In some humanized 3D6 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:H17 is occupied by L, H27 is occupied by Y, H29 is occupied by F, andH61 is occupied by E In some humanized 3D6 antibodies, positions H17,H27, H29, and H61 in the VH region are occupied by L, Y, F, and E,respectively.

In some humanized 3D6 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:H17 is occupied by L, H27 is occupied by Y, H29 is occupied by F, H61 isoccupied by E, H76 is occupied by N, and H82a is occupied by G.

In some humanized 3D6 antibodies, positions H17, H27, H29, H61, H76, andH82a in the VH region are occupied by L, Y, F, E, N, and G,respectively.

In some humanized 3D6 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:H12 is occupied by V, H17 is occupied by L, H24 is occupied by A, H43 isoccupied by Q, H48 is occupied by I, H83 is occupied by T, and H91 isoccupied by F. In some humanized 3D6 antibodies, positions H12, H17,H24, H43, H48, H83, and H91 in the VH region are occupied by V, L, A, Q,I, T, F, respectively.

In some humanized 3D6 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:H12 is occupied by V, H24 is occupied by A, H48 is occupied by I, H67 isoccupied b A, H80 is occupied by L, H83 is occupied by T, and H91 isoccupied by F. In some humanized 3D6 antibodies, positions H12, H24,H48, H67, H80, H83, and H91 in the VH region are occupied by V, A, I, A,L, T, and F, respectively.

In some humanized 3D6 antibodies, at least one of the followingpositions in the VH region is occupied by the amino acid as specified:H10 is occupied by E or D, H12 is occupied by K or V, H13 is occupied byK or R, H17 is occupied by T, L or S, H24 is occupied by V or A, H27 isoccupied by F or Y, H28 is occupied by N or T, H29 is occupied by I orF, H30 is occupied by K or T, H38 is occupied by Q or R, H40 is occupiedby A or R, H42 is occupied by G or E, H43 is occupied by K or Q, H48 isoccupied by M or I, H51 is occupied by V or I, H54 is occupied by N orD, H60 is occupied by D or A, H61 is occupied by P or E, H66 is occupiedby R or K, H67 is occupied by V or A, H76 is occupied by D or N, H80 isoccupied by M or L, H81 is occupied by E or Q, H82a is occupied by S orG, H83 is occupied by T or R, H91 is occupied by Y or F, H93 is occupiedby A or S, H102 is occupied by F or Y, H108 is occupied by T or L, H109is occupied by L or V.

In some humanized 3D6 antibodies, positions H12, H13, H17, H24, H38,H42, H43, H48, H66, H67, H76, H80, H81, H83, H91, and H93 in the VHregion are occupied by V, R, L, A, R, E, Q, I, K, A, N, L, Q, T, F, andS, respectively, as in hu3D6VHv1. In some humanized 3D6 antibodies,positions H38, H42, H43, H76, H83, and H93 in the VH region are occupiedby R, E, Q, N, T, and S, respectively, as in hu3D6VHv2. In somehumanized 3D6 antibodies, positions H12, H13, H17, H24, H38, H40, H42,H43, H48, H66, H67, H76, H80, H81, H82A, H83, H91, and H93 in the VHregion are occupied by V, R, L, A, R, R, E, Q, I, K, A, N, L, Q, G, T,F, and S, respectively, as in hu3D6VHv1b. In some humanized 3D6antibodies, positions H12, H24, H38, H40, H43, H48, H67, H80, H81, H82A,H83, H91, and H93 in the VH region are occupied by V, A, R, R, Q, I, A,L, Q, G, T, F, and S, respectively, as in hu3D6VHv1bA11. In somehumanized 3D6 antibodies, positions H12, H24, H28, H38, H40, H43, H48,H54, H60, H67, H80, H81, H82A, H83, H91, and H93 in the VH region areoccupied by V, A, T, R, R, Q, I, D, A, A, L, Q, G, T, F, and S,respectively, as in hu3D6VHv5. In some humanized 3D6 antibodies,positions H12, H24, H28, H38, H40, H48, H51, H54, H60, H67, H80, H82A,H83, H91, and H93 in the VH region are occupied by V, A, T, R, R, I, V,D, A, A, L, G, T, F, and S, respectively, as in hu3D6VHv1bA11B6G2. Insome humanized 3D6 antibodies, positions H12, H24, H28, H38, H40, H48,H54, H60, H67, H80, H82A, H83, H91, and H93 in the VH region areoccupied by V, A, T, R, R, I, D, A, A, L, G, T, F, and S, respectively,as in hu3D6VHv1bA11B6H3. In some humanized 3D6 antibodies, positionsH13, H17, H24, H29, H38, H40, H42, H43, H54, H61, H76, H80, H81, H82A,H83, H91, and H93 in the VH region are occupied by R, L, A, F, R, R, E,Q, N, E, N, L, Q, G, T, F, and S, respectively, as in hu3D6VHv1c. Insome humanized 3D6 antibodies, positions H13, H17, H24, H27, H28, H29,H30, H38, H40, H42, H43, H51, H54, H60, H61, H76, H80, H81, H82A, H83,H91, and H93 in the VH region are occupied by R, L, A, Y, T, F, T, R, R,E, Q, V, D, A, E, N, L, Q, G, T, F, and S, respectively, as inhu3D6VHv1d. In some humanized 3D6 antibodies, positions H10, H12, H17,H24, H28, H38, H40, H42, H43, H48, H54, H60, H61, H76, H80, H82A, H83,H91, H93, H108, and H109 in the VH region are occupied by D, V, L, A, T,R, R, E, Q, I, N, A, E, N, L, G, T, F, S, T, and L, respectively, as in,hu3D6VHv1e. In some humanized 3D6 antibodies, positions H10, H12, H17,H24, H28, H38, H40, H43, H48, H51, H54, H60, H61, H82A, H83, H91, H93,H102, H108, and H109 in the VH region are occupied by D, V, L, A, T, R,R, Q, I, V, D, A, E, G, T, F, S, Y, T, and L, respectively, as inhu3D6VHv1f. In some humanized 3D6 antibodies, positions H38 and H93 inthe VH region are occupied by R and S, respectively, as in hu3D6VHv3. Insome humanized 3D6 antibodies, positions H17, H27, H29, H38, H61, H76,H82A, and H93 in the VH region are occupied by L, Y, F, R, E, N, G, andS, respectively, as in hu3D6VHv3b. In some humanized 3D6 antibodies,positions H17, H27, H28, H29, H30, H38, H51, H54, H60, H61, H76, H82A,and H93 in the VH region are occupied by L, Y, T, F, T, R, V, D, A, E,N, G, and S, respectively, as in hu3D6VHv3c. In some humanized 3D6antibodies, positions H12, H38, H40, H48, H66, H67, H76, H80, H82A, H83,and H93 in the VH region are occupied by V, R, R, I, K, A, N, L, G, T,and S, respectively, as in hu3D6VHv4. In some humanized 3D6 antibodies,positions H12, H17, H27, H29, H38, H40, H61, H80, H82A, H83, and H93 inthe VH region are occupied by V, L, Y, F, R, R, E, L, G, T, and S,respectively, as in hu3D6VHv4b. In some humanized 3D6 antibodies,positions H12, H17, H27, H28, H29, H30, H38, H40, H51, H54, H60, H61,H80, H82A, H83, and H93 in the VH region are occupied by V, L, Y, T, F,T, R, R, V, D, A, E, L, G, T, and S, respectively, as in hu3D6VHv4c.

In some humanized 3D6 antibodies, at least one of the followingpositions in the VL region is occupied by the amino acid as specified:L36 is occupied by L, L37 is occupied by L, and L100 is occupied by G.In some humanized 3D6 antibodies, positions L36, L37, and L100 regionare occupied by L, L, and G, respectively.

In some humanized 3D6 antibodies, at least one of the followingpositions in the VL region is occupied by the amino acid as specified:L12 is occupied by S and L45 is occupied by K. In some humanized 13D6antibodies, positions L12 and L45 in the VL region are occupied by S andK, respectively.

In some humanized 3D6 antibodies, at least one of the followingpositions in the VL region is occupied by the amino acid as specified:L2 is V or I, L7 is S or T, L12 is P or S, L15 is L or I, L36 is L, L37is L, L45 is R or K, L60 is D or S, L100 is G.

In some humanized 3D6 antibodies, positions L12, L36, L37, L45, and L100in the VL region are occupied by S, L, L, K, and G, respectively, as inhu3D6VLv1. In some humanized 3D6 antibodies, positions L36, L37, andL100 in the VL region are occupied by L, L and G, respectively, as inhu3D6VLv2. In some humanized 3D6 antibodies, positions L36, L37, L60,and L100 in the VL region are occupied by L, L, S, and G, respectively,as in hu3D6VLv3. In some humanized 3D6 antibodies, positions L2, L7,L12, L15, L36, L37, L45, and L100 in the VL region are occupied by I, T,S, I, L, L, K, and G, respectively, as in hu3D6VLv4. In some humanized3D6 antibodies, positions L2, L7, L12, L15, L36, L37, L45, and L100 inthe VL region are occupied by V, S, P, L, L, L, R, and G, respectively.

In some humanized 3D6 antibodies, the variable heavy chain has 85%identity to human sequence. In some humanized 3D6 antibodies, thevariable light chain has 85% identity to human sequence. In somehumanized 3D6 antibodies, each of the variable heavy chain and variablelight chain has 85% identity to human germline sequence. In somehumanized 3D6 antibodies, the three heavy chain CDRs are as defined byKabat/Chothia Composite (SEQ ID NOs: 8, 9, and 10) and the three lightchain CDRs are as defined by Kabat/Chothia Composite (SEQ ID NOs: 12,13, and 14); provided that position H27 is occupied by F or Y, H28 isoccupied by N or T, position H29 is occupied by I or F, position H30 isoccupied by K or T, position H51 is occupied by I or V, position H54 isoccupied by N or D, position H60 is occupied by D or A, position H61 isoccupied by P or E, and position H102 is occupied by F or Y. In somehumanized 3D6 antibodies, Kabat/Chothia Composite CDR-H1 has an aminoacid sequence comprising SEQ ID NO: 42, SEQ ID NO:58, SEQ ID NO:69, orSEQ ID NO:60. In some humanized 3D6 antibodies, Kabat CDR-H2 has anamino acid sequence comprising SEQ ID NO: 43, SEQ ID NO:61, SEQ IDNO:62, SEQ ID NO:63, or SEQ ID NO:64. In some humanized 3D6 antibodies,Kabat CDR-H3 has an amino acid sequence comprising SEQ ID NO:65.

The CDR regions of such humanized antibodies can be identical orsubstantially identical to the CDR regions of 3D6, The CDR regions canbe defined by any conventional definition (e.g., Chothia, or compositeof Chothia and Kabat) but are preferably as defined by Kabat.

Variable regions framework positions are in accordance with Kabatnumbering unless otherwise stated. Other such variants typically differfrom the sequences of the exemplified Hu3D6 heavy and light chains by asmall number (e.g., typically no more than 1, 2, 3, 5, 10, or 15) ofreplacements, deletions or insertions. Such differences are usually inthe framework but can also occur in the CDRs.

A possibility for additional variation in humanized 3D6 variants isadditional backmutations in the variable region frameworks. Many of theframework residues not in contact with the CDRs in the humanized mAb canaccommodate substitutions of amino acids from the correspondingpositions of the donor mouse mAb or other mouse or human antibodies, andeven many potential CDR-contact residues are also amenable tosubstitution. Even amino acids within the CDRs may be altered, forexample, with residues found at the corresponding position of the humanacceptor sequence used to supply variable region frameworks. Inaddition, alternate human acceptor sequences can be used, for example,for the heavy and/or light chain. If different acceptor sequences areused, one or more of the backmutations recommended above may not beperformed because the corresponding donor and acceptor residues arealready the same without backmutations.

Preferably, replacements or backmutations in humanized 3D6 variants(whether or not conservative) have no substantial effect on the bindingaffinity or potency of the humanized mAb, that is, its ability to bindto tau.

The humanized 3D6 antibodies are further characterized by their abilityto bind both phosphorylated and unphosphorylated tau andmisfolded/aggregated forms of tau.

D. Chimeric and Veneered Antibodies

The invention further provides chimeric and veneered forms of non-humanantibodies, particularly the 3D6 antibodies of the examples.

A chimeric antibody is an antibody in which the mature variable regionsof light and heavy chains of a non-human antibody (e.g., a mouse) arecombined with human light and heavy chain constant regions. Suchantibodies substantially or entirely retain the binding specificity ofthe mouse antibody, and are about two-thirds human sequence. In anembodiment, a chimeric 3D6 antibody has a heavy chain amino acidsequence of SEQ ID NO: 72 and a light chain amino acid sequence of SEQID NO:73.

A veneered antibody is a type of humanized antibody that retains someand usually all of the CDRs and some of the non-human variable regionframework residues of a non-human antibody but replaces other variableregion framework residues that may contribute to B- or T-cell epitopes,for example exposed residues (Padlan, Mol. Immunol. 28:489, 1991) withresidues from the corresponding positions of a human antibody sequence.The result is an antibody in which the CDRs are entirely orsubstantially from a non-human antibody and the variable regionframeworks of the non-human antibody are made more human-like by thesubstitutions. Veneered forms of the 3D6 antibody are included in theinvention.

E. Human Antibodies

Human antibodies against tau or a fragment thereof (e.g., amino acidresidues 199-213 or 262-276 of SEQ ID NO:3, corresponding to amino acidresidues 257-271 or 320-334, respectively, of SEQ ID NO:1) are providedby a variety of techniques described below. Some human antibodies areselected by competitive binding experiments, by the phage display methodof Winter, above, or otherwise, to have the same epitope specificity asa particular mouse antibody, such as one of the mouse monoclonalantibodies described in the examples. Human antibodies can also bescreened for a particular epitope specificity by using only a fragmentof tau, such as a tau fragment containing only amino acid residues199-213 or 262-276 of SEQ ID NO:3 (corresponding to amino acid residues257-271 or 320-334, respectively, of SEQ ID NO:1) as the target antigen,and/or by screening antibodies against a collection of tau variants,such as tau variants containing various mutations within amino acidresidues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to amino acidresidues 257-271 or 320-334, respectively, of SEQ ID NO:1).

Methods for producing human antibodies include the trioma method ofOestberg et al., Hybridoma 2:361-367 (1983); Oestberg, U.S. Pat. No.4,634,664; and Engleman et al., U.S. Pat. No. 4,634,666, use oftransgenic mice including human immunoglobulin genes (see, e.g., Lonberget al., WO93/12227 (1993); U.S. Pat. Nos. 5,877,397; 5,874,299;5,814,318; 5,789,650; 5,770,429; 5,661,016; 5,633,425; 5,625,126;5,569,825; 5,545,806; Neuberger, Nat. Biotechnol. 14:826 (1996); andKucherlapati, WO 91/10741 (1991)) phage display methods (see, e.g.,Dower et al., WO 91/17271; McCafferty et al., WO 92/01047; U.S. Pat.Nos. 5,877,218; 5,871,907; 5,858,657; 5,837,242; 5,733,743; and5,565,332); and methods described in WO 2008/081008 (e.g., immortalizingmemory B cells isolated from humans, e.g., with EBV, screening fordesired properties, and cloning and expressing recombinant forms).

F. Selection of Constant Region

The heavy and light chain variable regions of chimeric, veneered orhumanized antibodies can be linked to at least a portion of a humanconstant region. The choice of constant region depends, in part, whetherantibody-dependent cell-mediated cytotoxicity, antibody dependentcellular phagocytosis and/or complement dependent cytotoxicity aredesired. For example, human isotypes IgG1 and IgG3 havecomplement-dependent cytotoxicity and human isotypes IgG2 and IgG4 donot. Human IgG1 and IgG3 also induce stronger cell mediated effectorfunctions than human IgG2 and IgG4. Light chain constant regions can belambda or kappa. Numbering conventions for constant regions include EUnumbering (Edelman, G. M. et al., Proc. Natl. Acad. USA, 63, 78-85(1969)), Kabat numbering (Kabat, Sequences of Proteins of ImmunologicalInterest (National Institutes of Health, Bethesda, Md., 1991, IMGTunique numbering (Lefranc M.-P. et al., IMGT unique numbering forimmunoglobulin and T cell receptor constant domains and Ig superfamilyC-like domains, Dev. Comp. Immunol., 29, 185-203 (2005), and IMGT exonnumbering (Lefranc, supra).

One or several amino acids at the amino or carboxy terminus of the lightand/or heavy chain, such as the C-terminal lysine of the heavy chain,may be missing or derivatized in a proportion or all of the molecules.Substitutions can be made in the constant regions to reduce or increaseeffector function such as complement-mediated cytotoxicity or ADCC (see,e.g., Winter et al., U.S. Pat. No. 5,624,821; Tso et al., U.S. Pat. No.5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006),or to prolong half-life in humans (see, e.g., Hinton et al., J. Biol.Chem. 279:6213, 2004). Exemplary substitutions include a Gln at position250 and/or a Leu at position 428 (EU numbering is used in this paragraphfor the constant region) for increasing the half-life of an antibody.Substitution at any or all of positions 234, 235, 236 and/or 237 reduceaffinity for Fcγ receptors, particularly FcγRI receptor (see, e.g., U.S.Pat. No. 6,624,821). An alanine substitution at positions 234, 235, and237 of human IgG1 can be used for reducing effector functions. Someantibodies have alanine substitution at positions 234, 235 and 237 ofhuman IgG1 for reducing effector functions. Optionally, positions 234,236 and/or 237 in human IgG2 are substituted with alanine and position235 with glutamine (see, e.g., U.S. Pat. No. 5,624,821). In someantibodies, a mutation at one or more of positions 241, 264, 265, 270,296, 297, 322, 329, and 331 by EU numbering of human IgG1 is used. Insome antibodies, a mutation at one or more of positions 318, 320, and322 by EU numbering of human IgG1 is used. In some antibodies, positions234 and/or 235 are substituted with alanine and/or position 329 issubstituted with glycine. In some antibodies, positions 234 and 235 aresubstituted with alanine. In some antibodies, the isotype is human IgG2or IgG4.

Antibodies can be expressed as tetramers containing two light and twoheavy chains, as separate heavy chains, light chains, as Fab, Fab′,F(ab′)2, and Fv, or as single chain antibodies in which heavy and lightchain mature variable domains are linked through a spacer.

Human constant regions show allotypic variation and isoallotypicvariation between different individuals, that is, the constant regionscan differ in different individuals at one or more polymorphicpositions. Isoallotypes differ from allotypes in that sera recognizingan isoallotype bind to a non-polymorphic region of a one or more otherisotypes. Thus, for example, another heavy chain constant region is ofIgG1 G1m3 with or without the C-terminal lysine. Reference to a humanconstant region includes a constant region with any natural allotype orany permutation of residues occupying positions in natural allotypes.

G. Expression of Recombinant Antibodies

A number of methods are known for producing chimeric and humanizedantibodies using an antibody-expressing cell line (e.g., hybridoma). Forexample, the immunoglobulin variable regions of antibodies can be clonedand sequenced using well known methods. In one method, the heavy chainvariable VH region is cloned by RT-PCR using mRNA prepared fromhybridoma cells. Consensus primers are employed to the VH region leaderpeptide encompassing the translation initiation codon as the 5′ primerand a g2b constant regions specific 3′ primer. Exemplary primers aredescribed in U.S. patent publication US 2005/0009150 by Schenk et al.(hereinafter “Schenk”). The sequences from multiple, independentlyderived clones can be compared to ensure no changes are introducedduring amplification. The sequence of the VH region can also bedetermined or confirmed by sequencing a VH fragment obtained by 5′ RACERT-PCR methodology and the 3′ g2b specific primer.

The light chain variable VL region can be cloned in an analogous manner.In one approach, a consensus primer set is designed for amplification ofVL regions using a 5′ primer designed to hybridize to the VL regionencompassing the translation initiation codon and a 3′ primer specificfor the Ck region downstream of the V-J joining region. In a secondapproach, 5′RACE RT-PCR methodology is employed to clone a VL encodingcDNA. Exemplary primers are described in Schenk, supra. The clonedsequences are then combined with sequences encoding human (or othernon-human species) constant regions.

In one approach, the heavy and light chain variable regions arere-engineered to encode splice donor sequences downstream of therespective VDJ or VJ junctions and are cloned into a mammalianexpression vector, such as pCMV-hγ1 for the heavy chain and pCMV-Mc1 forthe light chain. These vectors encode human γ1 and Ck constant regionsas exonic fragments downstream of the inserted variable region cassette.Following sequence verification, the heavy chain and light chainexpression vectors can be co-transfected into CHO cells to producechimeric antibodies. Conditioned media is collected 48 hourspost-transfection and assayed by western blot analysis for antibodyproduction or ELISA for antigen binding. The chimeric antibodies arehumanized as described above.

Chimeric, veneered, humanized, and human antibodies are typicallyproduced by recombinant expression. Recombinant polynucleotideconstructs typically include an expression control sequence operablylinked to the coding sequences of antibody chains, including naturallyassociated or heterologous expression control elements, such as apromoter. The expression control sequences can be promoter systems invectors capable of transforming or transfecting eukaryotic orprokaryotic host cells. Once the vector has been incorporated into theappropriate host, the host is maintained under conditions suitable forhigh level expression of the nucleotide sequences and the collection andpurification of the crossreacting antibodies.

These expression vectors are typically replicable in the host organismseither as episomes or as an integral part of the host chromosomal DNA.Commonly, expression vectors contain selection markers, e.g., ampicillinresistance or hygromycin resistance, to permit detection of those cellstransformed with the desired DNA sequences.

E. coli is one prokaryotic host useful for expressing antibodies,particularly antibody fragments. Microbes, such as yeast, are alsouseful for expression. Saccharomyces is a yeast host with suitablevectors having expression control sequences, an origin of replication,termination sequences, and the like as desired. Typical promotersinclude 3-phosphoglycerate kinase and other glycolytic enzymes.Inducible yeast promoters include, among others, promoters from alcoholdehydrogenase, isocytochrome C, and enzymes responsible for maltose andgalactose utilization.

Mammalian cells can be used for expressing nucleotide segments encodingimmunoglobulins or fragments thereof. See Winnacker, From Genes toClones, (VCH Publishers, N Y, 1987). A number of suitable host celllines capable of secreting intact heterologous proteins have beendeveloped, and include CHO cell lines, various COS cell lines, HeLacells, HEK293 cells, L cells, and non-antibody-producing myelomasincluding Sp2/0 and NS0. The cells can be nonhuman. Expression vectorsfor these cells can include expression control sequences, such as anorigin of replication, a promoter, an enhancer (Queen et al., Immunol.Rev. 89:49 (1986)), and necessary processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Expression control sequences caninclude promoters derived from endogenous genes, cytomegalovirus, SV40,adenovirus, bovine papillomavirus, and the like. See Co et al., J.Immunol. 148:1149 (1992).

Alternatively, antibody coding sequences can be incorporated intransgenes for introduction into the genome of a transgenic animal andsubsequent expression in the milk of the transgenic animal (see, e.g.,U.S. Pat. Nos. 5,741,957; 5,304,489; and 5,849,992). Suitable transgenesinclude coding sequences for light and/or heavy chains operably linkedwith a promoter and enhancer from a mammary gland specific gene, such ascasein or beta lactoglobulin.

The vectors containing the DNA segments of interest can be transferredinto the host cell by methods depending on the type of cellular host.For example, calcium chloride transfection is commonly utilized forprokaryotic cells, whereas calcium phosphate treatment, electroporation,lipofection, biolistics, or viral-based transfection can be used forother cellular hosts. Other methods used to transform mammalian cellsinclude the use of polybrene, protoplast fusion, liposomes,electroporation, and microinjection. For production of transgenicanimals, transgenes can be microinjected into fertilized oocytes or canbe incorporated into the genome of embryonic stem cells, and the nucleiof such cells transferred into enucleated oocytes.

Having introduced vector(s) encoding antibody heavy and light chainsinto cell culture, cell pools can be screened for growth productivityand product quality in serum-free media. Top-producing cell pools canthen be subjected of FACS-based single-cell cloning to generatemonoclonal lines. Specific productivities above 50 pg or 100 pg per cellper day, which correspond to product titers of greater than 7.5 g/Lculture, can be used. Antibodies produced by single cell clones can alsobe tested for turbidity, filtration properties, PAGE, IEF, UV scan,HP-SEC, carbohydrate-oligosaccharide mapping, mass spectrometry, andbinding assay, such as ELISA or Biacore. A selected clone can then bebanked in multiple vials and stored frozen for subsequent use.

Once expressed, antibodies can be purified according to standardprocedures of the art, including protein A capture, HPLC purification,column chromatography, gel electrophoresis and the like (see generally,Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

Methodology for commercial production of antibodies can be employed,including codon optimization, selection of promoters, selection oftranscription elements, selection of terminators, serum-free single cellcloning, cell banking, use of selection markers for amplification ofcopy number, CHO terminator, or improvement of protein titers (see,e.g., U.S. Pat. Nos. 5,786,464; 6,114,148; 6,063,598; 7,569,339;WO2004/050884; WO2008/012142; WO2008/012142; WO2005/019442;WO2008/107388; WO2009/027471; and U.S. Pat. No. 5,888,809).

IV. Active Immunogens

An agent used for active immunization serves to induce in a patient thesame types of antibody described in connection with passive immunizationabove. Agents used for active immunization can be the same types ofimmunogens used for generating monoclonal antibodies in laboratoryanimals, e.g., a peptide of 3-15 or 3-12 or 5-12, or 5-8 contiguousamino acids from a region of tau corresponding to residues 199-213 or262-276 of SEQ ID NO:3 (corresponding to residues 257-271 or 320-334,respectively, of SEQ ID NO:1), such as, for example, a peptide includingresidues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to residues257-271 or 320-334, respectively, of SEQ ID NO:1). For inducingantibodies binding to the same or overlapping epitope as 3D6, theepitope specificity of these antibodies can be mapped (e.g., by testingbinding to a series of overlapping peptides spanning tau). A fragment oftau consisting of or including or overlapping the epitope can then beused as an immunogen. Such fragments are typically used inunphosphorylated form.

The heterologous carrier and adjuvant, if used may be the same as usedfor generating monoclonal antibody, but may also be selected for betterpharmaceutical suitability for use in humans. Suitable carriers includeserum albumins, keyhole limpet hemocyanin, immunoglobulin molecules,thyroglobulin, ovalbumin, tetanus toxoid, or a toxoid from otherpathogenic bacteria, such as diphtheria (e.g., CRM197), E. coli,cholera, or H. pylori, or an attenuated toxin derivative. T cellepitopes are also suitable carrier molecules. Some conjugates can beformed by linking agents of the invention to an immunostimulatorypolymer molecule (e.g., tripalmitoyl-S-glycerine cysteine (Pam₃Cys),mannan (a mannose polymer), or glucan (αβ1→2 polymer)), cytokines (e.g.,IL-1, IL-1 alpha and β peptides, IL-2, γ-INF, IL-10, GM-CSF), andchemokines (e.g., MIP1-α and β, and RANTES). Immunogens may be linked tothe carriers with or without spacers amino acids (e.g., gly-gly).Additional carriers include virus-like particles. Virus-like particles(VLPs), also called pseudovirions or virus-derived particles, representsubunit structures composed of multiple copies of a viral capsid and/orenvelope protein capable of self-assembly into VLPs of defined sphericalsymmetry in vivo. (Powilleit, et al., (2007) PLoS ONE 2(5):e415.)Alternatively, peptide immunogens can be linked to at least oneartificial T-cell epitope capable of binding a large proportion of MHCClass II molecules, such as the pan DR epitope (“PADRE”). PADRE isdescribed in U.S. Pat. No. 5,736,142, WO 95/07707, and Alexander J etal, Immunity, 1:751-761 (1994). Active immunogens can be presented inmultimeric form in which multiple copies of an immunogen and/or itscarrier are presented as a single covalent molecule.

Fragments are often administered with pharmaceutically acceptableadjuvants. The adjuvant increases the titer of induced antibodies and/orthe binding affinity of induced antibodies relative to the situation ifthe peptide were used alone. A variety of adjuvants can be used incombination with an immunogenic fragment of tau to elicit an immuneresponse. Preferred adjuvants augment the intrinsic response to animmunogen without causing conformational changes in the immunogen thataffect the qualitative form of the response. Preferred adjuvants includealuminum salts, such as aluminum hydroxide and aluminum phosphate, 3De-O-acylated monophosphoryl lipid A (MPL™) (see GB 2220211 (RIBIImmunoChem Research Inc., Hamilton, Mont., now part of Corixa).Stimulon™ QS-21 is a triterpene glycoside or saponin isolated from thebark of the Quillaja Saponaria Molina tree found in South America (seeKensil et al., in Vaccine Design: The Subunit and Adjuvant Approach(eds. Powell & Newman, Plenum Press, N Y, 1995); U.S. Pat. No.5,057,540), (Aquila BioPharmaceuticals, Framingham, Mass.; nowAntigenics, Inc., New York, N.Y.). Other adjuvants are oil in wateremulsions (such as squalene or peanut oil), optionally in combinationwith immune stimulants, such as monophosphoryl lipid A (see Stoute etal., N. Engl. J. Med. 336, 86-91 (1997)), pluronic polymers, and killedmycobacteria. Ribi adjuvants are oil-in-water emulsions. Ribi contains ametabolizable oil (squalene) emulsified with saline containing Tween 80.Ribi also contains refined mycobacterial products which act asimmunostimulants and bacterial monophosphoryl lipid A. Another adjuvantis CpG (WO 98/40100). Adjuvants can be administered as a component of atherapeutic composition with an active agent or can be administeredseparately, before, concurrently with, or after administration of thetherapeutic agent.

Analogs of natural fragments of tau that induce antibodies against taucan also be used. For example, one or more or all L-amino acids can besubstituted with D amino acids in such peptides. Also the order of aminoacids can be reversed (retro peptide). Optionally a peptide includes allD-amino acids in reverse order (retro-inverso peptide). Peptides andother compounds that do not necessarily have a significant amino acidsequence similarity with tau peptides but nevertheless serve as mimeticsof tau peptides and induce a similar immune response. Anti-idiotypicantibodies against monoclonal antibodies to tau as described above canalso be used. Such anti-Id antibodies mimic the antigen and generate animmune response to it (see Essential Immunology, Roit ed., BlackwellScientific Publications, Palo Alto, Calif. 6th ed., p. 181).

Peptides (and optionally a carrier fused to the peptide) can also beadministered in the form of a nucleic acid encoding the peptide andexpressed in situ in a patient. A nucleic acid segment encoding animmunogen is typically linked to regulatory elements, such as a promoterand enhancer that allow expression of the DNA segment in the intendedtarget cells of a patient. For expression in blood cells, as isdesirable for induction of an immune response, promoter and enhancerelements from light or heavy chain immunoglobulin genes or the CMV majorintermediate early promoter and enhancer are suitable to directexpression. The linked regulatory elements and coding sequences areoften cloned into a vector. Antibodies can also be administered in theform of nucleic acids encoding the antibody heavy and/or light chains.If both heavy and light chains are present, the chains are preferablylinked as a single chain antibody. Antibodies for passive administrationcan also be prepared e.g., by affinity chromatography from sera ofpatients treated with peptide immunogens.

The DNA can be delivered in naked form (i.e., without colloidal orencapsulating materials). Alternatively a number of viral vector systemscan be used including retroviral systems (see, e.g., Lawrie and Tumin,Cur. Opin. Genet. Develop. 3, 102-109 (1993)); adenoviral vectors {see,e.g., Bett et al, J. Virol. 67, 591 1 (1993)); adeno-associated virusvectors {see, e.g., Zhou et al., J. Exp. Med. 179, 1867 (1994)), viralvectors from the pox family including vaccinia virus and the avian poxviruses, viral vectors from the alpha virus genus such as those derivedfrom Sindbis and Semliki Forest Viruses (see, e.g., Dubensky et al., J.Virol. 70, 508-519 (1996)), Venezuelan equine encephalitis virus (seeU.S. Pat. No. 5,643,576) and rhabdoviruses, such as vesicular stomatitisvirus (see WO 96/34625) and papillomaviruses (Ohe et al., Human GeneTherapy 6, 325-333 (1995); Woo et al, WO 94/12629 and Xiao & Brandsma,Nucleic Acids. Res. 24, 2630-2622 (1996)).

DNA encoding an immunogen, or a vector containing the same, can bepackaged into liposomes. Suitable lipids and related analogs aredescribed by U.S. Pat. Nos. 5,208,036, 5,264,618, 5,279,833, and5,283,185. Vectors and DNA encoding an immunogen can also be adsorbed toor associated with particulate carriers, examples of which includepolymethyl methacrylate polymers and polylactides andpoly(lactide-co-glycolides), (see, e.g., McGee et al., J. Micro Encap.1996).

H. Antibody Screening Assays

Antibodies can be initially screened for the intended bindingspecificity as described above. Active immunogens can likewise bescreened for capacity to induce antibodies with such bindingspecificity. In this case, an active immunogen is used to immunize alaboratory animal and the resulting sera tested for the appropriatebinding specificity.

Antibodies having the desired binding specificity can then be tested incellular and animal models. The cells used for such screening arepreferentially neuronal cells. A cellular model of tau pathology hasbeen reported in which neuroblastoma cells are transfected with afour-repeat domain of tau, optionally with a mutation associated withtau pathology (e.g., delta K280, see Khlistunova, Current AlzheimerResearch 4, 544-546 (2007)). In another model, tau is induced in theneuroblastoma N2a cell line by the addition of doxycyclin. The cellmodels enable one to study the toxicity of tau to cells in the solubleor aggregated state, the appearance of tau aggregates after switching ontau gene expression, the dissolution of tau aggregates after switchingthe gene expression off again, and the efficiency of antibodies ininhibiting formation of tau aggregates or disaggregating them.

Antibodies or active immunogens can also be screened in transgenicanimal models of diseases associated with tau. Such transgenic animalscan include a tau transgene (e.g., any of the human isoforms) andoptionally a human APP transgene among others, such as a kinase thatphosphorylates tau, ApoE, presenilin or alpha synuclein. Such transgenicanimals are disposed to develop at least one sign or symptom of adisease associated with tau.

An exemplary transgenic animal is the K3 line of mice (Itner et al.,Proc. Natl. Acad. Sci. USA 105(41):15997-6002 (2008)). These mice have ahuman tau transgene with a K 369 I mutation (the mutation is associatedwith Pick's disease) and a Thy 1.2 promoter. This model shows a rapidcourse of neurodegeneration, motor deficit and degeneration of afferentfibers and cerebellar granule cells. Another exemplary animal is theJNPL3 line of mice. These mice have a human tau transgene with a P301Lmutation (the mutation is associated with frontotemporal dementia) and aThy 1.2 promoter (Taconic, Germantown, N.Y., Lewis, et al., Nat Genet.25:402-405 (2000)). These mice have a more gradual course ofneurodegeneration. The mice develop neurofibrillary tangles in severalbrain regions and spinal cord, which is hereby incorporated by referencein its entirety). This is an excellent model to study the consequencesof tangle development and for screening therapy that may inhibit thegeneration of these aggregates. Another advantage of these animals isthe relatively early onset of pathology. In the homozygous line,behavioral abnormalities associated with tau pathology can be observedat least as early as 3 months, but the animals remain relatively healthyat least until 8 months of age. In other words, at 8 months, the animalsambulate, feed themselves, and can perform the behavioral taskssufficiently well to allow the treatment effect to be monitored. Activeimmunization of these mice for 6-13 months with—AI wI KLH-PHF-1generated titers of about 1,000 and showed fewer neurofibrillarytangles, less pSer422, and reduced weight loss relative to untreatedcontrol ice.

The activity of antibodies or active agents can be assessed by variouscriteria including reduction in amount of total tau or phosphorylatedtau, reduction in other pathological characteristics, such as amyloiddeposits of Aβ, and inhibition or delay or behavioral deficits. Activeimmunogens can also be tested for induction of antibodies in the sera.Both passive and active immunogens can be tested for passage ofantibodies across the blood brain barrier into the brain of a transgenicanimal. Antibodies or fragments inducing an antibody can also be testedin non-human primates that naturally or through induction developsymptoms of diseases characterized by tau. Tests on an antibody oractive agent are usually performed in conjunction with a control inwhich a parallel experiment is conduct except that the antibody oractive agent is absent (e.g., replaced by vehicle). Reduction, delay orinhibition of signs or symptoms disease attributable to an antibody oractive agent under test can then be assessed relative to the control.

V. Patients Amenable to Treatment

The presence of neurofibrillary tangles has been found in severaldiseases including Alzheimer's disease, Down's syndrome, mild cognitiveimpairment, primary age-related tauopathy, postencephaliticparkinsonism, posttraumatic dementia or dementia pugilistica, Pick'sdisease, type C Niemann-Pick disease, supranuclear palsy, frontotemporaldementia, frontotemporal lobar degeneration, argyrophilic grain disease,globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonismdementia complex of Guam, corticobasal degeneration (CBD), dementia withLewy bodies, Lewy body variant of Alzheimer disease (LBVAD), andprogressive supranuclear palsy (PSP). The present regimes can also beused in treatment or prophylaxis of any of these diseases. Because ofthe widespread association between neurological diseases and conditionsand tau, the present regimes can be used in treatment or prophylaxis ofany subject showing elevated levels of tau or phosphorylated tau (e.g.,in the CSF) compared with a mean value in individuals withoutneurological disease. The present regimes can also be used in treatmentor prophylaxis of neurological disease in individuals having a mutationin tau associated with neurological disease. The present methods areparticularly suitable for treatment or prophylaxis of Alzheimer'sdisease, and especially in patients.

Patients amenable to treatment include individuals at risk of diseasebut not showing symptoms, as well as patients presently showingsymptoms. Patients at risk of disease include those having a knowngenetic risk of disease. Such individuals include those having relativeswho have experienced this disease, and those whose risk is determined byanalysis of genetic or biochemical markers. Genetic markers of riskinclude mutations in tau, such as those discussed above, as well asmutations in other genes associated with neurological disease. Forexample, the ApoE4 allele in heterozygous and even more so in homozygousform is associated with risk of Alzheimer's disease. Other markers ofrisk of Alzheimer's disease include mutations in the APP gene,particularly mutations at position 717 and positions 670 and 671referred to as the Hardy and Swedish mutations respectively, mutationsin the presenilin genes, PS1 and PS2, a family history of AD,hypercholesterolemia or atherosclerosis. Individuals presently sufferingfrom Alzheimer's disease can be recognized by PET imaging, fromcharacteristic dementia, as well as the presence of risk factorsdescribed above. In addition, a number of diagnostic tests are availablefor identifying individuals who have AD. These include measurement ofCSF tau or phospho-tau and Aβ42 levels. Elevated tau or phospho-tau anddecreased Aβ42 levels signify the presence of AD. Some mutationsassociated with Parkinson's disease. Ala30Pro or Ala53, or mutations inother genes associated with Parkinson's disease such as leucine-richrepeat kinase, PARKS. Individuals can also be diagnosed with any of theneurological diseases mentioned above by the criteria of the DSM IV TR.

In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,30). Usually, however, it is not necessary to begin treatment until apatient reaches 40, 50, 60 or 70 years of age. Treatment typicallyentails multiple dosages over a period of time. Treatment can bemonitored by assaying antibody levels over time. If the response falls,a booster dosage is indicated. In the case of potential Down's syndromepatients, treatment can begin antenatally by administering therapeuticagent to the mother or shortly after birth.

I. Nucleic Acids

The invention further provides nucleic acids encoding any of the heavyand light chains described above (e.g., SEQ ID NOs: 7, 11, 15-19, andSEQ ID NOs: 20-23, SEQ ID NOs: 46-57, and SEQ ID NO:66). Optionally,such nucleic acids further encode a signal peptide and can be expressedwith the signal peptide linked to the constant region. Coding sequencesof nucleic acids can be operably linked with regulatory sequences toensure expression of the coding sequences, such as a promoter, enhancer,ribosome binding site, transcription termination signal, and the like.The nucleic acids encoding heavy and light chains can occur in isolatedform or can be cloned into one or more vectors. The nucleic acids can besynthesized by, for example, solid state synthesis or PCR of overlappingoligonucleotides. Nucleic acids encoding heavy and light chains can bejoined as one contiguous nucleic acid, e.g., within an expressionvector, or can be separate, e.g., each cloned into its own expressionvector.

J. Conjugated Antibodies

Conjugated antibodies that specifically bind to antigens, such as tau,are useful in detecting the presence of tau; monitoring and evaluatingthe efficacy of therapeutic agents being used to treat patientsdiagnosed with Alzheimer's disease, Down's syndrome, mild cognitiveimpairment, primary age-related tauopathy, postencephaliticparkinsonism, posttraumatic dementia or dementia pugilistica, Pick'sdisease, type C Niemann-Pick disease, supranuclear palsy, frontotemporaldementia, frontotemporal lobar degeneration, argyrophilic grain disease,globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonismdementia complex of Guam, corticobasal degeneration (CBD), dementia withLewy bodies, Lewy body variant of Alzheimer disease (LBVAD), orprogressive supranuclear palsy (PSP); inhibiting or reducing aggregationof tau; inhibiting or reducing tau fibril formation; reducing orclearing tau deposits; stabilizing non-toxic conformations of tau; ortreating or effecting prophylaxis of Alzheimer's disease, Down'ssyndrome, mild cognitive impairment, primary age-related tauopathy,postencephalitic parkinsonism, posttraumatic dementia or dementiapugilistica, Pick's disease, type C Niemann-Pick disease, supranuclearpalsy, frontotemporal dementia, frontotemporal lobar degeneration,argyrophilic grain disease, globular glial tauopathy, amyotrophiclateral sclerosis/parkinsonism dementia complex of Guam, corticobasaldegeneration (CBD), dementia with Lewy bodies, Lewy body variant ofAlzheimer disease (LBVAD), or progressive supranuclear palsy (PSP) in apatient. For example, such antibodies can be conjugated with othertherapeutic moieties, other proteins, other antibodies, and/ordetectable labels. See WO 03/057838; U.S. Pat. No. 8,455,622. Suchtherapeutic moieties can be any agent that can be used to treat, combat,ameliorate, prevent, or improve an unwanted condition or disease in apatient, such as Alzheimer's disease, Down's syndrome, mild cognitiveimpairment, primary age-related tauopathy, postencephaliticparkinsonism, posttraumatic dementia or dementia pugilistica, Pick'sdisease, type C Niemann-Pick disease, supranuclear palsy, frontotemporaldementia, frontotemporal lobar degeneration, argyrophilic grain disease,globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonismdementia complex of Guam, corticobasal degeneration (CBD), dementia withLewy bodies, Lewy body variant of Alzheimer disease (LBVAD), orprogressive supranuclear palsy (PSP).

Conjugated therapeutic moieties can include cytotoxic agents, cytostaticagents, neurotrophic agents, neuroprotective agents, radiotherapeuticagents, immunomodulators, or any biologically active agents thatfacilitate or enhance the activity of the antibody. A cytotoxic agentcan be any agent that is toxic to a cell. A cytostatic agent can be anyagent that inhibits cell proliferation. A neurotrophic agent can be anyagent, including chemical or proteinaceous agents, that promotes neuronmaintenance, growth, or differentiation. A neuroprotective agent can beagent, including chemical or proteinaceous agents, that protects neuronsfrom acute insult or degenerative processes. An immunomodulator can beany agent that stimulates or inhibits the development or maintenance ofan immunologic response. A radiotherapeutic agent can be any molecule orcompound that emits radiation. If such therapeutic moieties are coupledto a tau-specific antibody, such as the antibodies described herein, thecoupled therapeutic moieties will have a specific affinity fortau-related disease-affected cells over normal cells. Consequently,administration of the conjugated antibodies directly targets cancercells with minimal damage to surrounding normal, healthy tissue. Thiscan be particularly useful for therapeutic moieties that are too toxicto be administered on their own. In addition, smaller quantities of thetherapeutic moieties can be used.

Some such antibodies can be modified to act as immunotoxins. See, e.g.,U.S. Pat. No. 5,194,594. For example, ricin, a cellular toxin derivedfrom plants, can be coupled to antibodies by using the bifunctionalreagents S-acetylmercaptosuccinic anhydride for the antibody andsuccinimidyl 3-(2-pyridyldithio)propionate for ricin. See Pietersz etal., Cancer Res. 48(16):4469-4476 (1998). The coupling results in lossof B-chain binding activity of ricin, while impairing neither the toxicpotential of the A-chain of ricin nor the activity of the antibody.Similarly, saporin, an inhibitor of ribosomal assembly, can be coupledto antibodies via a disulfide bond between chemically insertedsulfhydryl groups. See Polito et al., Leukemia 18:1215-1222 (2004).

Some such antibodies can be linked to radioisotopes. Examples ofradioisotopes include, for example, yttrium⁹⁰ (90Y), indium¹¹¹ (111In),¹³¹I, ⁹⁹mTc, radiosilver-111, radiosilver-199, and Bismuth²¹³. Linkageof radioisotopes to antibodies may be performed with conventionalbifunction chelates. For radiosilver-111 and radiosilver-199 linkage,sulfur-based linkers may be used. See Hazra et al., Cell Biophys.24-25:1-7 (1994). Linkage of silver radioisotopes may involve reducingthe immunoglobulin with ascorbic acid. For radioisotopes such as 111Inand 90Y, ibritumomab tiuxetan can be used and will react with suchisotopes to form 111In-ibritumomab tiuxetan and 90Y-ibritumomabtiuxetan, respectively. See Witzig, Cancer Chemother. Pharmacol., 48Suppl 1:S91-S95 (2001).

Some such antibodies can be linked to other therapeutic moieties. Suchtherapeutic moieties can be, for example, cytotoxic, cytostatic,neurotrophic, or neuroprotective. For example, antibodies can beconjugated with toxic chemotherapeutic drugs such as maytansine,geldanamycin, tubulin inhibitors such as tubulin binding agents (e.g.,auristatins), or minor groove binding agents such as calicheamicin.Other representative therapeutic moieties include agents known to beuseful for treatment, management, or amelioration of Alzheimer'sdisease, Down's syndrome, mild cognitive impairment, primary age-relatedtauopathy, postencephalitic parkinsonism, posttraumatic dementia ordementia pugilistica, Pick's disease, type C Niemann-Pick disease,supranuclear palsy, frontotemporal dementia, frontotemporal lobardegeneration, argyrophilic grain disease, globular glial tauopathy,amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam,corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy bodyvariant of Alzheimer disease (LBVAD), or progressive supranuclear palsy(PSP).

Antibodies can also be coupled with other proteins. For example,antibodies can be coupled with Fynomers. Fynomers are small bindingproteins (e.g., 7 kDa) derived from the human Fyn SH3 domain. They canbe stable and soluble, and they can lack cysteine residues and disulfidebonds. Fynomers can be engineered to bind to target molecules with thesame affinity and specificity as antibodies. They are suitable forcreating multi-specific fusion proteins based on antibodies. Forexample, Fynomers can be fused to N-terminal and/or C-terminal ends ofantibodies to create bi- and tri-specific FynomAbs with differentarchitectures. Fynomers can be selected using Fynomer libraries throughscreening technologies using FACS, Biacore, and cell-based assays thatallow efficient selection of Fynomers with optimal properties. Examplesof Fynomers are disclosed in Grabulovski et al., J. Biol. Chem.282:3196-3204 (2007); Bertschinger et al., Protein Eng. Des. Sel.20:57-68 (2007); Schlatter et al., MAbs. 4:497-508 (2011); Banner etal., Acta. Crystallogr. D. Biol. Crystallo 69(Pt6):1124-1137 (2013); andBrack et al., Mol. Cancer Ther. 13:2030-2039 (2014).

The antibodies disclosed herein can also be coupled or conjugated to oneor more other antibodies (e.g., to form antibody heteroconjugates). Suchother antibodies can bind to different epitopes within tau or can bindto a different target antigen.

Antibodies can also be coupled with a detectable label. Such antibodiescan be used, for example, for diagnosing Alzheimer's disease, Down'ssyndrome, mild cognitive impairment, primary age-related tauopathy,postencephalitic parkinsonism, posttraumatic dementia or dementiapugilistica, Pick's disease, type C Niemann-Pick disease, supranuclearpalsy, frontotemporal dementia, frontotemporal lobar degeneration,argyrophilic grain disease, globular glial tauopathy, amyotrophiclateral sclerosis/parkinsonism dementia complex of Guam, corticobasaldegeneration (CBD), dementia with Lewy bodies, Lewy body variant ofAlzheimer disease (LBVAD), or progressive supranuclear palsy (PSP),and/or for assessing efficacy of treatment. Such antibodies areparticularly useful for performing such determinations in subjectshaving or being susceptible to Alzheimer's disease, Down's syndrome,mild cognitive impairment, primary age-related tauopathy,postencephalitic parkinsonism, posttraumatic dementia or dementiapugilistica, Pick's disease, type C Niemann-Pick disease, supranuclearpalsy, frontotemporal dementia, frontotemporal lobar degeneration,argyrophilic grain disease, globular glial tauopathy, amyotrophiclateral sclerosis/parkinsonism dementia complex of Guam, corticobasaldegeneration (CBD), dementia with Lewy bodies, Lewy body variant ofAlzheimer disease (LBVAD), or progressive supranuclear palsy (PSP), orin appropriate biological samples obtained from such subjects.Representative detectable labels that may be coupled or linked to anantibody include various enzymes, such as horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;prosthetic groups, such streptavidin/biotin and avidin/biotin;fluorescent materials, such as umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; luminescent materials, such as luminol;bioluminescent materials, such as luciferase, luciferin, and aequorin;radioactive materials, such as radiosilver-111, radiosilver-199,Bismuth²¹³, iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon (¹⁴C), sulfur (⁵S),tritium (³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In), technetium (⁹⁹Tc),thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum(⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm,¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge,⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, and¹¹⁷Tin; positron emitting metals using various positron emissiontomographies; nonradioactive paramagnetic metal ions; and molecules thatare radiolabelled or conjugated to specific radioisotopes.

Linkage of radioisotopes to antibodies may be performed withconventional bifunction chelates. For radiosilver-111 andradiosilver-199 linkage, sulfur-based linkers may be used. See Hazra etal., Cell Biophys. 24-25:1-7 (1994). Linkage of silver radioisotopes mayinvolve reducing the immunoglobulin with ascorbic acid. Forradioisotopes such as 111In and 90Y, ibritumomab tiuxetan can be usedand will react with such isotopes to form 111In-ibritumomab tiuxetan and90Y-ibritumomab tiuxetan, respectively. See Witzig, Cancer Chemother.Pharmacol., 48 Suppl 1:S91-S95 (2001).

Therapeutic moieties, other proteins, other antibodies, and/ordetectable labels may be coupled or conjugated, directly or indirectlythrough an intermediate (e.g., a linker), to an antibody of theinvention. See e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy,” in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery,” inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review,” in Monoclonal Antibodies 84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy,” inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985); and Thorpe et al., Immunol.Rev., 62:119-58 (1982). Suitable linkers include, for example, cleavableand non-cleavable linkers. Different linkers that release the coupledtherapeutic moieties, proteins, antibodies, and/or detectable labelsunder acidic or reducing conditions, on exposure to specific proteases,or under other defined conditions can be employed.

VI. Pharmaceutical Compositions and Methods of Use

In prophylactic applications, an antibody or agent for inducing anantibody or a pharmaceutical composition the same is administered to apatient susceptible to, or otherwise at risk of a disease (e.g.,Alzheimer's disease) in regime (dose, frequency and route ofadministration) effective to reduce the risk, lessen the severity, ordelay the onset of at least one sign or symptom of the disease. Inparticular, the regime is preferably effective to inhibit or delay tauor phospho-tau and paired filaments formed from it in the brain, and/orinhibit or delay its toxic effects and/or inhibit/or delay developmentof behavioral deficits. In therapeutic applications, an antibody oragent to induce an antibody is administered to a patient suspected of,or already suffering from a disease (e.g., Alzheimer's disease) in aregime (dose, frequency and route of administration) effective toameliorate or at least inhibit further deterioration of at least onesign or symptom of the disease. In particular, the regime is preferablyeffective to reduce or at least inhibit further increase of levels oftau, phosphor-tau, or paired filaments formed from it, associatedtoxicities and/or behavioral deficits.

A regime is considered therapeutically or prophylactically effective ifan individual treated patient achieves an outcome more favorable thanthe mean outcome in a control population of comparable patients nottreated by methods of the invention, or if a more favorable outcome isdemonstrated in treated patients versus control patients in a controlledclinical trial (e.g., a phase II, phase II/III or phase III trial) atthe p<0.05 or 0.01 or even 0.001 level.

Effective doses of vary depending on many different factors, such asmeans of administration, target site, physiological state of thepatient, whether the patient is an ApoE carrier, whether the patient ishuman or an animal, other medications administered, and whethertreatment is prophylactic or therapeutic.

Exemplary dosage ranges for antibodies are from about 0.01 to 60 mg/kg,or from about 0.1 to 3 mg/kg or 0.15-2 mg/kg or 0.15-1.5 mg/kg, ofpatient body weight. Antibody can be administered such doses daily, onalternative days, weekly, fortnightly, monthly, quarterly, or accordingto any other schedule determined by empirical analysis. An exemplarytreatment entails administration in multiple dosages over a prolongedperiod, for example, of at least six months. Additional exemplarytreatment regimes entail administration once per every two weeks or oncea month or once every 3 to 6 months.

The amount of an agent for active administration varies from 0.1-500 μgper patient and more usually from 1-100 or 1-10 μg per injection forhuman administration. The timing of injections can vary significantlyfrom once a day, to once a year, to once a decade. A typical regimenconsists of an immunization followed by booster injections at timeintervals, such as 6 week intervals or two months. Another regimenconsists of an immunization followed by booster injections 1, 2 and 12months later. Another regimen entails an injection every two months forlife. Alternatively, booster injections can be on an irregular basis asindicated by monitoring of immune response.

Antibodies or agents for inducing antibodies are preferably administeredvia a peripheral route (i.e., one in which an administered or inducedantibody crosses the blood brain barrier to reach an intended site inthe brain. Routes of administration include topical, intravenous, oral,subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal,intranasal, intraocular, or intramuscular. Preferred routes foradministration of antibodies are intravenous and subcutaneous. Preferredroutes for active immunization are subcutaneous and intramuscular. Thistype of injection is most typically performed in the arm or leg muscles.In some methods, agents are injected directly into a particular tissuewhere deposits have accumulated, for example intracranial injection.

Pharmaceutical compositions for parenteral administration are preferablysterile and substantially isotonic and manufactured under GMPconditions. Pharmaceutical compositions can be provided in unit dosageform (i.e., the dosage for a single administration). Pharmaceuticalcompositions can be formulated using one or more physiologicallyacceptable carriers, diluents, excipients or auxiliaries. Theformulation depends on the route of administration chosen. Forinjection, antibodies can be formulated in aqueous solutions, preferablyin physiologically compatible buffers such as Hank's solution, Ringer'ssolution, or physiological saline or acetate buffer (to reducediscomfort at the site of injection). The solution can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively antibodies can be in lyophilized form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The present regimes can be administered in combination with anotheragent effective in treatment or prophylaxis of the disease beingtreated. For example, in the case of Alzheimer's disease, the presentregimes can be combined with immunotherapy against Aβ (WO/2000/072880),cholinesterase inhibitors or memantine or in the case of Parkinson'sdisease immunotherapy against alpha synuclein WO/2008/103472, Levodopa,dopamine agonists, COMT inhibitors, MAO-B inhibitors, Amantadine, oranticholinergic agents.

Antibodies are administered in an effective regime meaning a dosage,route of administration and frequency of administration that delays theonset, reduces the severity, inhibits further deterioration, and/orameliorates at least one sign or symptom of a disorder being treated. Ifa patient is already suffering from a disorder, the regime can bereferred to as a therapeutically effective regime. If the patient is atelevated risk of the disorder relative to the general population but isnot yet experiencing symptoms, the regime can be referred to as aprophylactically effective regime. In some instances, therapeutic orprophylactic efficacy can be observed in an individual patient relativeto historical controls or past experience in the same patient. In otherinstances, therapeutic or prophylactic efficacy can be demonstrated in apreclinical or clinical trial in a population of treated patientsrelative to a control population of untreated patients.

Exemplary dosages for an antibody are 0.1-60 mg/kg (e.g., 0.5, 3, 10,30, or 60 mg/kg), or 0.5-5 mg/kg body weight (e.g., 0.5, 1, 2, 3, 4 or 5mg/kg) or 10-4000 mg or 10-1500 mg as a fixed dosage. The dosage dependson the condition of the patient and response to prior treatment, if any,whether the treatment is prophylactic or therapeutic and whether thedisorder is acute or chronic, among other factors.

Administration can be parenteral, intravenous, oral, subcutaneous,intra-arterial, intracranial, intrathecal, intraperitoneal, topical,intranasal or intramuscular. Some antibodies can be administered intothe systemic circulation by intravenous or subcutaneous administration.Intravenous administration can be, for example, by infusion over aperiod such as 30-90 min.

The frequency of administration depends on the half-life of the antibodyin the circulation, the condition of the patient and the route ofadministration among other factors. The frequency can be daily, weekly,monthly, quarterly, or at irregular intervals in response to changes inthe patient's condition or progression of the disorder being treated. Anexemplary frequency for intravenous administration is between weekly andquarterly over a continuous cause of treatment, although more or lessfrequent dosing is also possible. For subcutaneous administration, anexemplary dosing frequency is daily to monthly, although more or lessfrequent dosing is also possible.

The number of dosages administered depends on whether the disorder isacute or chronic and the response of the disorder to the treatment. Foracute disorders or acute exacerbations of a chronic disorder, between 1and 10 doses are often sufficient. Sometimes a single bolus dose,optionally in divided form, is sufficient for an acute disorder or acuteexacerbation of a chronic disorder. Treatment can be repeated forrecurrence of an acute disorder or acute exacerbation. For chronicdisorders, an antibody can be administered at regular intervals, e.g.,weekly, fortnightly, monthly, quarterly, every six months for at least1, 5 or 10 years, or the life of the patient.

A. Diagnostics and Monitoring Methods

In Vivo Imaging, Diagnostic Methods, and Optimizing Immunotherapy

The invention provides methods of in vivo imaging tau protein deposits(e.g., neurofibrillary tangles and tau inclusions) in a patient. Themethods work by administering a reagent, such as antibody that binds tau(e.g., a mouse, humanized, chimeric or veneered 3D6 antibody), to thepatient and then detecting the agent after it has bound. Antibodiesbinding to an epitope of tau within amino acid residues 199-213 or262-276 of SEQ ID NO:3 (corresponding to amino acid residues 257-271 or320-334, respectively, of SEQ ID NO:1) are preferred. In some methods,the antibody binds to an epitope within amino acid residues 199-213 ofSEQ ID NO:3 (corresponding to amino acid residues 257-271 of SEQ IDNO:1), or within amino acids 262-276 of SEQ ID NO:3 (corresponding toamino acid residues 320-334 of SEQ ID NO:1). A clearing response to theadministered antibodies can be avoided or reduced by using antibodyfragments lacking a full-length constant region, such as Fabs. In somemethods, the same antibody can serve as both a treatment and diagnosticreagent.

Diagnostic reagents can be administered by intravenous injection intothe body of the patient, or directly into the brain by intracranialinjection or by drilling a hole through the skull. The dosage of reagentshould be within the same ranges as for treatment methods. Typically,the reagent is labeled, although in some methods, the primary reagentwith affinity for tau is unlabeled and a secondary labeling agent isused to bind to the primary reagent. The choice of label depends on themeans of detection. For example, a fluorescent label is suitable foroptical detection. Use of paramagnetic labels is suitable fortomographic detection without surgical intervention. Radioactive labelscan also be detected using positron emission tomography (PET) orsingle-photon emission computed tomography (SPECT).

The methods of in vivo imaging of tau protein deposits are useful todiagnose or confirm diagnosis of a tauopathy, such as Alzheimer'sdisease, frontotemporal lobar degeneration, progressive supranuclearpalsy and Pick's disease, or susceptibility to such a disease. Forexample, the methods can be used on a patient presenting with symptomsof dementia. If the patient has abnormal neurofibrillary tangles, thenthe patient is likely suffering from Alzheimer's disease. Alternatively,if the patient has abnormal tau inclusions, then depending on thelocation of the inclusions, the patient may be suffering fromfrontotemporal lobar degeneration. The methods can also be used onasymptomatic patients. Presence of abnormal tau protein depositsindicates susceptibility to future symptomatic disease. The methods arealso useful for monitoring disease progression and/or response totreatment in patients who have been previously diagnosed with atau-related disease.

Diagnosis can be performed by comparing the number, size, and/orintensity of labeled loci, to corresponding baseline values. The baseline values can represent the mean levels in a population of undiseasedindividuals. Baseline values can also represent previous levelsdetermined in the same patient. For example, baseline values can bedetermined in a patient before beginning tau immunotherapy treatment,and measured values thereafter compared with the baseline values. Adecrease in values relative to baseline signals a positive response totreatment.

In some patients, diagnosis of a tauopathy may be aided by performing aPET scan. A PET scan can be performed using, for example, a conventionalPET imager and auxiliary equipment. The scan typically includes one ormore regions of the brain known in general to be associated with tauprotein deposits and one or more regions in which few if any depositsare generally present to serve as controls.

The signal detected in a PET scan can be represented as amultidimensional image. The multidimensional image can be in twodimensions representing a cross-section through the brain, in threedimensions, representing the three dimensional brain, or in fourdimensions representing changes in the three dimensional brain overtime. A color scale can be used with different colors indicatingdifferent amounts of label and, inferentially, tau protein depositdetected. The results of the scan can also be presented numerically,with numbers relating to the amount of label detected and consequentlyamount of tau protein deposits. The label present in a region of thebrain known to be associated with deposits for a particular tauopathy(e.g., Alzheimer's disease) can be compared with the label present in aregion known not to be associated with deposits to provide a ratioindicative of the extent of deposits within the former region. For thesame radiolabeled ligand, such ratios provide a comparable measure oftau protein deposits and changes thereof between different patients.

In some methods, a PET scan is performed concurrent with or in the samepatient visit as an MRI or CAT scan. An MM or CAT scan provides moreanatomical detail of the brain than a PET scan. However, the image froma PET scan can be superimposed on an MRI or CAT scan image moreprecisely indicating the location of PET ligand and inferentially taudeposits relative to anatomical structures in the brain. Some machinescan perform both PET scanning and MRI or CAT scanning without thepatient changing positions between the scans facilitatingsuperimposition of images.

Suitable PET ligands include radiolabeled antibodies of the invention(e.g., a mouse, humanized, chimeric or veneered 3D6 antibody). Theradioisotope used can be, for example, C¹¹, N¹³, O¹⁵, F¹⁸, or I¹²³. Theinterval between administering the PET ligand and performing the scancan depend on the PET ligand and particularly its rate of uptake andclearing into the brain, and the half-life of its radiolabel.

PET scans can also be performed as a prophylactic measure inasymptomatic patients or in patients who have symptoms of mild cognitiveimpairment but have not yet been diagnosed with a tauopathy but are atelevated risk of developing a tauopathy. For asymptomatic patients,scans are particularly useful for individuals considered at elevatedrisk of tauopathy because of a family history, genetic or biochemicalrisk factors, or mature age. Prophylactic scans can commence forexample, at a patient age between 45 and 75 years. In some patients, afirst scan is performed at age 50 years.

Prophylactic scans can be performed at intervals of for example, betweensix months and ten years, preferably between 1-5 years. In somepatients, prophylactic scans are performed annually. If a PET scanperformed as a prophylactic measure indicates abnormally high levels oftau protein deposits, immunotherapy can be commenced and subsequent PETscans performed as in patients diagnosed with a tauopathy. If a PETscanned performed as a prophylactic measure indicates levels of tauprotein deposits within normal levels, further PET scans can performedat intervals of between six months and 10 years, and preferably 1-5years, as before, or in response to appearance of signs and symptoms ofa tauopathy or mild cognitive impairment. By combining prophylacticscans with administration of tau-directed immunotherapy if and when anabove normal level of tau protein deposits is detected, levels of tauprotein deposits can be reduced to, or closer to, normal levels, or atleast inhibited from increasing further, and the patient can remain freeof the tauopathy for a longer period than if not receiving prophylacticscans and tau-directed immunotherapy (e.g., at least 5, 10, 15 or 20years, or for the rest of the patient's life).

Normal levels of tau protein deposits can be determined by the amount ofneurofibrillary tangles or tau inclusions in the brains of arepresentative sample of individuals in the general population who havenot been diagnosed with a particular tauopathy (e.g., Alzheimer'sdisease) and are not considered at elevated risk of developing suchdisease (e.g., a representative sample of disease-free individuals under50 years of age). Alternatively, a normal level can be recognized in anindividual patient if the PET signal according to the present methods ina region of the brain in which tau protein deposits are known to developis not different (within the accuracy of measurement) from the signalfrom a region of the brain in which it is known that such deposits donot normally develop. An elevated level in an individual can berecognized by comparison to the normal levels (e.g., outside mean andvariance of a standard deviation) or simply from an elevated signalbeyond experimental error in a region of the brain associated with tauprotein deposits compared with a region not known to be associated withdeposits. For purposes of comparing the levels of tau protein depositsin an individual and population, the tau protein deposits shouldpreferably be determined in the same region(s) of the brain, theseregions including at least one region in which tau protein depositsassociated with a particular tauopathy (e.g., Alzheimer's disease) areknown to form. A patient having an elevated level of tau proteindeposits is a candidate for commencing immunotherapy.

After commencing immunotherapy, a decrease in the level of tau proteindeposits can be first seen as an indication that the treatment is havingthe desired effect. The observed decrease can be, for example, in therange of 1-100%, 1-50%, or 1-25% of the baseline value. Such effects canbe measured in one or more regions of the brain in which deposits areknown to form or can be measured from an average of such regions. Thetotal effect of treatment can be approximated by adding the percentagereduction relative to baseline to the increase in tau protein depositsthat would otherwise occur in an average untreated patient.

Maintenance of tau protein deposits at an approximately constant levelor even a small increase in tau protein deposits can also be anindication of response to treatment albeit a suboptimal response. Suchresponses can be compared with a time course of levels of tau proteindeposits in patients with a particular tauopathy (e.g., Alzheimer'sdisease) that did not receive treatment, to determine whether theimmunotherapy is having an effect in inhibiting further increases of tauprotein deposits.

Monitoring of changes in tau protein deposits allows adjustment of theimmunotherapy or other treatment regime in response to the treatment.PET monitoring provides an indication of the nature and extent ofresponse to treatment. Then a determination can be made whether toadjust treatment and if desired treatment can be adjusted in response tothe PET monitoring. PET monitoring thus allows for tau-directedimmunotherapy or other treatment regime to be adjusted before otherbiomarkers, MRI or cognitive measures have detectably responded. Asignificant change means that comparison of the value of a parameterafter treatment relative to basement provides some evidence thattreatment has or has not resulted in a beneficial effect. In someinstances, a change of values of a parameter in a patient itselfprovides evidence that treatment has or has not resulted in a beneficialeffect. In other instances, the change of values, if any, in a patient,is compared with the change of values, if any, in a representativecontrol population of patients not undergoing immunotherapy. Adifference in response in a particular patient from the normal responsein the control patient (e.g., mean plus variance of a standarddeviation) can also provide evidence that an immunotherapy regime is oris not achieving a beneficial effect in a patient.

In some patients, monitoring indicates a detectable decline in tauprotein deposits but that the level of tau protein deposits remainsabove normal. In such patients, if there are no unacceptable sideeffects, the treatment regime can be continued as is or even increasedin frequency of administration and/or dose if not already at the maximumrecommended dose.

If the monitoring indicates levels of tau protein deposits in a patienthave already been reduced to normal, or near-normal, levels of tauprotein deposits, the immunotherapy regime can be adjusted from one ofinduction (i.e., that reduces the level of tau protein deposits) to oneof maintenance (i.e., that maintains tau protein deposits at anapproximately constant level). Such a regime can be affected by reducingthe dose and or frequency of administering immunotherapy.

In other patients, monitoring can indicate that immunotherapy is havingsome beneficial effect but a suboptimal effect. An optimal effect can bedefined as a percentage reduction in the level of tau protein depositswithin the top half or quartile of the change in tau protein deposits(measured or calculated over the whole brain or representative region(s)thereof in which tau protein deposits are known to form) experienced bya representative sample of tauopathy patients undergoing immunotherapyat a given time point after commencing therapy. A patient experiencing asmaller decline or a patient whose tau protein deposits remains constantor even increases, but to a lesser extent than expected in the absenceof immunotherapy (e.g., as inferred from a control group of patients notadministered immunotherapy) can be classified as experiencing a positivebut suboptimal response. Such patients can optionally be subject to anadjustment of regime in which the dose and or frequency ofadministration of an agent is increased.

In some patients, tau protein deposits may increase in similar orgreater fashion to tau deposits in patients not receiving immunotherapy.If such increases persist over a period of time, such as 18 months or 2years, even after any increase in the frequency or dose of agents,immunotherapy can if desired be discontinued in favor of othertreatments.

The foregoing description of diagnosing, monitoring, and adjustingtreatment for tauopathies has been largely focused on using PET scans.However, any other technique for visualizing and/or measuring tauprotein deposits that is amenable to the use of tau antibodies of theinvention (e.g., a mouse, humanized, chimeric or veneered 3D6 antibody)can be used in place of PET scans to perform such methods.

Also provided are methods of detecting an immune response against tau ina patient suffering from or susceptible to diseases associated with tau.The methods can be used to monitor a course of therapeutic andprophylactic treatment with the agents provided herein. The antibodyprofile following passive immunization typically shows an immediate peakin antibody concentration followed by an exponential decay. Without afurther dose, the decay approaches pretreatment levels within a periodof days to months depending on the half-life of the antibodyadministered. For example, the half-life of some human antibodies is ofthe order of 20 days.

In some methods, a baseline measurement of antibody to tau in thesubject is made before administration, a second measurement is made soonthereafter to determine the peak antibody level, and one or more furthermeasurements are made at intervals to monitor decay of antibody levels.When the level of antibody has declined to baseline or a predeterminedpercentage of the peak less baseline (e.g., 50%, 25% or 10%),administration of a further dose of antibody is administered. In somemethods, peak or subsequent measured levels less background are comparedwith reference levels previously determined to constitute a beneficialprophylactic or therapeutic treatment regime in other subjects. If themeasured antibody level is significantly less than a reference level(e.g., less than the mean minus one or, preferably, two standarddeviations of the reference value in a population of subjects benefitingfrom treatment) administration of an additional dose of antibody isindicated.

Also provided are methods of detecting tau in a subject, for example, bymeasuring tau in a sample from a subject or by in vivo imaging of tau ina subject. Such methods are useful to diagnose or confirm diagnosis ofdiseases associated with tau, or susceptibility thereto. The methods canalso be used on asymptomatic subjects. The presence of tau indicatessusceptibility to future symptomatic disease. The methods are alsouseful for monitoring disease progression and/or response to treatmentin subjects who have been previously diagnosed with Alzheimer's disease,Down's syndrome, mild cognitive impairment, primary age-relatedtauopathy, postencephalitic parkinsonism, posttraumatic dementia ordementia pugilistica, Pick's disease, type C Niemann-Pick disease,supranuclear palsy, frontotemporal dementia, frontotemporal lobardegeneration, argyrophilic grain disease, globular glial tauopathy,amyotrophic lateral sclerosis/parkinsonism dementia complex of Guam,corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy bodyvariant of Alzheimer disease (LBVAD), or progressive supranuclear palsy(PSP).

Biological samples obtained from a subject having, suspected of having,or at risk of having Alzheimer's disease, Down's syndrome, mildcognitive impairment, primary age-related tauopathy, postencephaliticparkinsonism, posttraumatic dementia or dementia pugilistica, Pick'sdisease, type C Niemann-Pick disease, supranuclear palsy, frontotemporaldementia, frontotemporal lobar degeneration, argyrophilic grain disease,globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonismdementia complex of Guam, corticobasal degeneration (CBD), dementia withLewy bodies, Lewy body variant of Alzheimer disease (LBVAD), orprogressive supranuclear palsy (PSP) can be contacted with theantibodies disclosed herein to assess the presence of tau. For example,levels of tau in such subjects may be compared to those present inhealthy subjects. Alternatively, levels of tau in such subjectsreceiving treatment for the disease may be compared to those of subjectswho have not been treated for Alzheimer's disease, Down's syndrome, mildcognitive impairment, primary age-related tauopathy, postencephaliticparkinsonism, posttraumatic dementia or dementia pugilistica, Pick'sdisease, type C Niemann-Pick disease, supranuclear palsy, frontotemporaldementia, frontotemporal lobar degeneration, argyrophilic grain disease,globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonismdementia complex of Guam, corticobasal degeneration (CBD), dementia withLewy bodies, Lewy body variant of Alzheimer disease (LBVAD), orprogressive supranuclear palsy (PSP). Some such tests involve a biopsyof tissue obtained from such subjects. ELISA assays may also be usefulmethods, for example, for assessing tau in fluid samples.

VII. Kits

The invention further provides kits (e.g., containers) comprising anantibody disclosed herein and related materials, such as instructionsfor use (e.g., package insert). The instructions for use may contain,for example, instructions for administration of the antibody andoptionally one or more additional agents. The containers of antibody maybe unit doses, bulk packages (e.g., multi-dose packages), or sub-unitdoses.

Package insert refers to instructions customarily included in commercialpackages of therapeutic products that contain information about theindications, usage, dosage, administration, contraindications and/orwarnings concerning the use of such therapeutic products

Kits can also include a second container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It can also include other materials desirable from acommercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

VIII. Other Applications

The antibodies can be used for detecting tau, or fragments thereof, inthe context of clinical diagnosis or treatment or in research. Forexample, the antibodies can be used to detect the presence of tau in abiological sample as an indication that the biological sample comprisestau deposits. Binding of the antibodies to the biological sample can becompared to binding of the antibodies to a control sample. The controlsample and the biological sample can comprise cells of the same tissueorigin. Control samples and biological samples can be obtained from thesame individual or different individuals and on the same occasion or ondifferent occasions. If desired, multiple biological samples andmultiple control samples are evaluated on multiple occasions to protectagainst random variation independent of the differences between thesamples. A direct comparison can then be made between the biologicalsample(s) and the control sample(s) to determine whether antibodybinding (i.e., the presence of tau) to the biological sample(s) isincreased, decreased, or the same relative to antibody binding to thecontrol sample(s). Increased binding of the antibody to the biologicalsample(s) relative to the control sample(s) indicates the presence oftau in the biological sample(s). In some instances, the increasedantibody binding is statistically significant. Optionally, antibodybinding to the biological sample is at least 1.5-fold, 2-fold, 3-fold,4-fold, 5-fold, 10-fold, 20-fold, or 100-fold higher than antibodybinding to the control sample.

In addition, the antibodies can be used to detect the presence of thetau in a biological sample to monitor and evaluate the efficacy of atherapeutic agent being used to treat a patient diagnosed withAlzheimer's disease, Down's syndrome, mild cognitive impairment, primaryage-related tauopathy, postencephalitic parkinsonism, posttraumaticdementia or dementia pugilistica, Pick's disease, type C Niemann-Pickdisease, supranuclear palsy, frontotemporal dementia, frontotemporallobar degeneration, argyrophilic grain disease, globular glialtauopathy, amyotrophic lateral sclerosis/parkinsonism dementia complexof Guam, corticobasal degeneration (CBD), dementia with Lewy bodies,Lewy body variant of Alzheimer disease (LBVAD), or progressivesupranuclear palsy (PSP). A biological sample from a patient diagnosedwith Alzheimer's disease, Down's syndrome, mild cognitive impairment,primary age-related tauopathy, postencephalitic parkinsonism,posttraumatic dementia or dementia pugilistica, Pick's disease, type CNiemann-Pick disease, supranuclear palsy, frontotemporal dementia,frontotemporal lobar degeneration, argyrophilic grain disease, globularglial tauopathy, amyotrophic lateral sclerosis/parkinsonism dementiacomplex of Guam, corticobasal degeneration (CBD), dementia with Lewybodies, Lewy body variant of Alzheimer disease (LBVAD), or progressivesupranuclear palsy (PSP) is evaluated to establish a baseline for thebinding of the antibodies to the sample (i.e., a baseline for thepresence of the tau in the sample) before commencing therapy with thetherapeutic agent. In some instances, multiple biological samples fromthe patient are evaluated on multiple occasions to establish both abaseline and measure of random variation independent of treatment. Atherapeutic agent is then administered in a regime. The regime mayinclude multiple administrations of the agent over a period of time.Optionally, binding of the antibodies (i.e., presence of tau) isevaluated on multiple occasions in multiple biological samples from thepatient, both to establish a measure of random variation and to show atrend in response to immunotherapy. The various assessments of antibodybinding to the biological samples are then compared. If only twoassessments are made, a direct comparison can be made between the twoassessments to determine whether antibody binding (i.e., presence oftau) has increased, decreased, or remained the same between the twoassessments. If more than two measurements are made, the measurementscan be analyzed as a time course starting before treatment with thetherapeutic agent and proceeding through the course of therapy. Inpatients for whom antibody binding to biological samples has decreased(i.e., the presence of tau), it can be concluded that the therapeuticagent was effective in treating the Alzheimer's disease, Down'ssyndrome, mild cognitive impairment, primary age-related tauopathy,postencephalitic parkinsonism, posttraumatic dementia or dementiapugilistica, Pick's disease, type C Niemann-Pick disease, supranuclearpalsy, frontotemporal dementia, frontotemporal lobar degeneration,argyrophilic grain disease, globular glial tauopathy, amyotrophiclateral sclerosis/parkinsonism dementia complex of Guam, corticobasaldegeneration (CBD), dementia with Lewy bodies, Lewy body variant ofAlzheimer disease (LBVAD), or progressive supranuclear palsy (PSP) inthe patient. The decrease in antibody binding can be statisticallysignificant. Optionally, binding decreases by at least 1%, 2%, 3%, 4%,5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.Assessment of antibody binding can be made in conjunction with assessingother signs and symptoms of Alzheimer's disease, Down's syndrome, mildcognitive impairment, primary age-related tauopathy, postencephaliticparkinsonism, posttraumatic dementia or dementia pugilistica, Pick'sdisease, type C Niemann-Pick disease, supranuclear palsy, frontotemporaldementia, frontotemporal lobar degeneration, argyrophilic grain disease,globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonismdementia complex of Guam, corticobasal degeneration (CBD), dementia withLewy bodies, Lewy body variant of Alzheimer disease (LBVAD), orprogressive supranuclear palsy (PSP).

The antibodies can also be used as research reagents for laboratoryresearch in detecting tau, or fragments thereof. In such uses,antibodies can be labeled with fluorescent molecules, spin-labeledmolecules, enzymes, or radioisotopes, and can be provided in the form ofkit with all the necessary reagents to perform the detection assay. Theantibodies can also be used to purify tau, or binding partners of tau,e.g., by affinity chromatography.

All patent filings, websites, other publications, accession numbers andthe like cited above or below are incorporated by reference in theirentirety for all purposes to the same extent as if each individual itemwere specifically and individually indicated to be so incorporated byreference. If different versions of a sequence are associated with anaccession number at different times, the version associated with theaccession number at the effective filing date of this application ismeant. The effective filing date means the earlier of the actual filingdate or filing date of a priority application referring to the accessionnumber if applicable. Likewise if different versions of a publication,website or the like are published at different times, the version mostrecently published at the effective filing date of the application ismeant unless otherwise indicated. Any feature, step, element,embodiment, or aspect of the invention can be used in combination withany other unless specifically indicated otherwise. Although the presentinvention has been described in some detail by way of illustration andexample for purposes of clarity and understanding, it will be apparentthat certain changes and modifications may be practiced within the scopeof the appended claims.

EXAMPLES Example 1. Identification of Tau Monoclonal Antibodies

Monoclonal antibodies against tau were generated as follows.Immunizations were performed with either recombinant N-terminallyHis-tagged 383 a.a. human tau (4R0N), containing a P301S mutation[immunogen A] or recombinant 383 a.a. human tau (4R0N), containing aP301S mutation, lacking an N-terminal His-tag [immunogen B]. Immunogenswere emulsified in RIBI adjuvant.

Five week old female Balb/c mice were intraperitoneally immunized with25 μg of immunogen A on day 0, and 10 μg of immunogen A each on days 7,14, 21, 27, 34, 48, 55, and 62. Mice were immunized with 10 μg ofimmunogen B on days 76 and 90. On days 43 and 98, mice were bled andtitered against immunogen A; on day 101 the animals with highest titerswere boosted with a terminal immunization of 50 μg immunogen B, whichwas delivered ½ intraperitoneally and ½ intravenously. Fused hybridomaswere screened via ELISA against both immunogens, and positives with thehighest signal were epitope mapped (see Example 2). 3D6 reacted topeptides corresponding to amino acid residues 199-213 of SEQ ID NO:3 andto amino acid residues. 262-276 of SEQ ID NO:3 (following numbering ofthe longest CNS isoform of tau, this corresponds to amino acid residues257-271 of SEQ ID NO:1 and to amino acid residues 320-334 of SEQ IDNO:1).

Example 2. Epitope Mapping of Antibody 3D6

A range of overlapping biotinylated peptides spanning the entire 383aa4R0N human tau protein were used for mapping the murine 3D6 antibody.Additional peptides were used to model potential post-translationalmodifications of the C- and N-terminal ends of the protein.

Biotinylated peptides were bound to separate wells of astreptavidin-coated ELISA plate. The plate was blocked and treated withmurine 3D6, followed by incubation with a horseradishperoxidase-conjugated anti-mouse antibody. After thorough washing, OPDwas applied to the plate and allowed to develop. The plate was read at450 nm absorbance. Background subtraction was performed with absorbancevalues from wells containing no primary antibody, and a threshold forpositive binding was set to 0.2 absorbance units. Positive binding wasdetected for the peptide spanning amino acid residues 199-213 (of SEQ IDNO:3), and amino acid residues 262-276 (of SEQ ID NO:3). Using thenumbering of the full-length 4R2N human tau protein (441 amino acids)these peptides correspond to amino acid residues 257-271 (of SEQ IDNO:1) and 320-334 (of SEQ ID NO:1). (FIG. 1 )

Example 3. Design of Humanized 3D6 Antibodies

The starting point or donor antibody for humanization was the mouseantibody 3D6. The heavy chain variable amino acid sequence of maturem3D6 is provided as SEQ ID NO:7. The light chain variable amino acidsequence of mature m3D6 is provided as SEQ ID NO:11. The heavy chainKabat/Chothia Composite CDR1, CDR2, and CDR3 amino acid sequences areprovided as SEQ ID NOs:8-10, respectively. The light chain Kabat CDR1,CDR2, and CDR3 amino acid sequences are provided as SEQ ID NOs 12-14respectively. Kabat numbering is used throughout.

The variable kappa (Vk) of 3D6 belongs to mouse Kabat subgroup 2 whichcorresponds to human Kabat subgroup 2 and the variable heavy (Vh) tomouse Kabat subgroup 2c which corresponds to human Kabat subgroup 1[Kabat E. A., et al., (1991), Sequences of Proteins of ImmunologicalInterest, Fifth Edition. NIH Publication No. 91-3242]. 16 residueChothia CDR-L1 belongs to canonical class 4, 7 residue Chothia CDR-L2 toclass 1, 9 residue Chothia CDR-L3 to class 1 in Vk [Martin A. C, andThornton J. M. (1996) J. Mol. Biol. 263:800-15. [Martin & Thornton,1996]. 10 residue Chothia CDR-H1 belongs to class 1, 17 residue ChothiaCDR-H2 to class 2 [Martin & Thornton, 1996]]. CDR-H3 has no canonicalclasses. A search was made over the protein sequences in the PDBdatabase [Deshpande N, et al., (2005) Nucleic Acids Res. 33: D233-7.] tofind structures which would provide a rough structural model of 3D6. Tobuild up a Fv model of 3D6, a structure of an antibody binding to anepitope that participates in the PrPC to PrPSc conformation change (pdbcode 1CR9; SEQ ID NO: 70) [Kanyo, Z. F., et al. (1999). J. Mol. Biol.293: 855-863] with a resolution of 2.0A was used. It retained the samecanonical structure for the loops as 3D6. Following 2015 INN antibodyhumanization rule [Jones, T. D., et al, (2016), The INNs and outs ofantibody nonproprietary names. mAbs doi: 10.1080/19420862.2015.1114320],a search of IMGT database allowed selection of suitable human germlineframeworks into which to graft the murine CDRs. For Vk, a human kappalight chain with IMGT #IGKV2-30*02 (human germline VH sequence) and IMGT#IGKJ2*01 (human germline VJ sequence) were chosen. IMGT #IGKV2-30*02has the same canonical classes for CDR-L1, CDR-L2 and L3 as mouse 3D6.IMGT #IGKV2-30*02 and IMGT #IGKJ2*01 belong to human kappa subgroup 2.Human VL acceptor AAZ09048.1 was also applied. For Vh, human heavy chainwith IMGT #IGHV1-69-2*01 (human germline VH sequence) and IMGT #IGKJ1*01(human germline VJ sequence) were chosen, which both belong to humanheavy chain subgroup 1. IMGT #IGHV1-69-2*01 shares the canonical form ofmouse 3D6 CDR-H1 and H2. For VH design 1 and 2, human VH acceptorBAC01986.1 from a search of the non-redundant protein sequence databasefrom NCBI was also applied (following the previous INN humanizationrule).

Following 2015 INN antibody humanization rule [Jones, T. D., et al,(2016), The INNs and outs of antibody nonproprietary names. mAbs doi:10.1080/19420862.2015.1114320], a search of IMGT database allowedselection of suitable human germline frameworks into which to graft themurine CDRs. Heavy and light chain variant sequences resulting fromantibody humanization process were further aligned to human germ linesequences using IMGT Domain GapAlign tool to assess the humanness of theheavy and light chain as outlined by WHO INN committee guidelines.(WHO-INN: International nonproprietary names (INN) for biological andbiotechnological substances (a review) (Internet) 2014. Available from:World Health Organization website. Residues were changed to align withcorresponding human germ line sequence, where possible, to enhancehumanness.

17 humanized heavy chain variable region variants and 4 humanized lightchain variable region variants were constructed containing differentpermutations of substitutions hu3D6VHv1 (SEQ ID NO:15); hu3D6VHv2 (SEQID NO:16); hu3D6VHv1b (SEQ ID NO:17); hu3D6VHv1bA11 (SEQ ID NO:18);hu3D6VHv5 (SEQ ID NO:19); hu3D6VHv1bA11B6G2 (SEQ ID NO:46);hu3D6VHv1bA11B6H3 (SEQ ID NO:47); hu3D6VHv1c (SEQ ID NO:48); hu3D6VHv1d(SEQ ID NO:49); hu3D6VHv1e (SEQ ID NO:50); hu3D6VHv1f (SEQ ID NO:51);hu3D6VHv3 (SEQ ID NO:52); hu3D6VHv3b (SEQ ID NO:53); hu3D6VHv3c (SEQ IDNO:54); hu3D6VHv4 (SEQ ID NO:55); hu3D6VHv4b (SEQ ID NO:56); andhu3D6VHv4c (SEQ ID NO:57); and hu3D6VLv1, hu3D6VLv2, hu3D6VLv3, andhu3D6VLv4 (SEQ ID NOs:20-23, respectively) (Tables 3 and 4). Theexemplary humanized Vk and Vh designs, with backmutations and othermutations based on selected human frameworks, are shown in Tables 3 and4, respectively. The bolded areas in Tables 3 and 4 indicate the CDRs asdefined by Kabat/Chothia Composite. SEQ ID NOs: 15-19, SEQ ID NOs:20-23, SEQ ID NOs: 46-57 contain backmutations and other mutations asshown in Table 5. The amino acids at positions in hu3D6VHv1, hu3D6VHv2,hu3D6VHv1b, hu3D6VHv1bA11, hu3D6VHv5, hu3D6VHv1bA11B6G2,hu3D6VHv1bA11B6H3, hu3D6VHv1c, hu3D6VHv1d, hu3D6VHv1e, hu3D6VHv1f,hu3D6VHv3, hu3D6VHv3b, hu3D6VHv3c, hu3D6VHv4, hu3D6VHv4b, and hu3D6VHv4care listed in Table 6. The amino acids at positions in hu3D6VLv1,hu3D6VLv2, hu3D6VLv3, and hu3D6VLv4 at are listed in Table 7. Thepercentage humanness for humanized VH chains hu3D6VHv1, hu3D6VHv2,hu3D6VHv1b, hu3D6VHv1bA11, hu3D6VHv5, hu3D6VHv1bA11B6G2,hu3D6VHv1bA11B6H3, hu3D6VHv1c, hu3D6VHv1d, hu3D6VHv1e, hu3D6VHv1f,hu3D6VHv3, hu3D6VHv3b, hu3D6VHv3c, hu3D6VHv4, hu3D6VHv4b, and hu3D6VHv4c(SEQ ID NOs: 15-19, 46-57 respectively) and humanized VL chainshu3D6VLv1, hu3D6VLv2, hu3D6VLv3, and hu3D6VLv4 (SEQ ID NOs:20-23,respectively) is shown in Table 8.

TABLE 3 Hu VH Acceptor Hu VH FR Acc. # Acceptor IMGT# FR Acc. # MurineIGHV1-69- IMGT# Kabat Linear FR 3D6 VH 2*01 IGHJ1*01 h3D6VHv1 h3D6VHv2h3D6VHv1b h3D6VHv1bA11 residue residue or (SEQ ID (SEQ ID (SEQ ID (SEQID (SEQ ID (SEQ ID (SEQ ID # # CDR NO: 7) NO: 25) NO: 26) NO: 15) NO:16) NO: 17) NO: 18)  1 1 Fr1 E E E E E E  2 2 Fr1 V V V V V V  3 3 Fr1 QQ Q Q Q Q  4 4 Fr1 L L L L L L  5 5 Fr1 Q V V V V V  6 6 Fr1 Q Q Q Q Q Q 7 7 Fr1 S S S S S S  8 8 Fr1 G G G G G G  9 9 Fr1 A A A A A A  10 10Fr1 D E E E E E  11 11 Fr1 L V V V V V  12 12 Fr1 V K V K V V  13 13 Fr1R K R K R K  14 14 Fr1 P P P P P P  15 15 Fr1 G G G G G G  16 16 Fr1 A AA A A A  17 17 Fr1 L T L S L T  18 18 Fr1 V V V V V V  19 19 Fr1 K K K KK K  20 20 Fr1 L I V V I I  21 21 Fr1 S S S S S S  22 22 Fr1 C C C C C C 23 23 Fr1 K K K K K K  24 24 Fr1 A V A V A A  25 25 Fr1 S S S S S S  2626 Fr1 G G G G G G  27 27 Fr1 F Y F F F F  28 28 Fr1 N T N N N N  29 29Fr1 I F I I I I  30 30 Fr1 K T K K K K  31 31 CDR-H1 D D D D D D  32 32CDR-H1 Y Y Y Y Y Y  33 33 CDR-H1 Y Y Y Y Y Y  34 34 CDR-H1 L M L L L L 35 35 CDR-H1 H H H H H H  35A CDR-H1  35B CDR-H1  36 36 Fr2 W W W W W W 37 37 Fr2 V V V V V V  38 38 Fr2 R Q R R R R  39 39 Fr2 Q Q Q Q Q Q  4040 Fr2 R A A A R R  41 41 Fr2 P P P P P P  42 42 Fr2 E G E E E G  43 43Fr2 Q K Q Q Q Q  44 44 Fr2 G G G G G G  45 45 Fr2 L L L L L L  46 46 Fr2E E E E E E  47 47 Fr2 W W W W W W  48 48 Fr2 I M I M I I  49 49 Fr2 G GG G G G  50 50 CDR-H2 W L W W W W  51 51 CDR-H2 I V I I I I  52 52CDR-H2 D D D D D D  52A 53 CDR-H2 P P P P P P  52B  52C  53 54 CDR-H2 EE E E E E  54 55 CDR-H2 N D N N N N  55 56 CDR-H2 G G G G G G  56 57CDR-H2 D E D D D D  57 58 CDR-H2 T T T T T T  58 59 CDR-H2 V I V V V V 59 60 CDR-H2 Y Y Y Y Y Y  60 61 CDR-H2 D A D D D D  61 62 CDR-H2 P E PP P P  62 63 Fr3 K K K K K K  63 64 Fr3 F F F F F F  64 65 Fr3 Q Q Q Q QQ  65 66 Fr3 G G G G G G  66 67 Fr3 K R K R K R  67 68 Fr3 A V A V A A 68 69 Fr3 T T T T T T  69 70 Fr3 I I I I I I  70 71 Fr3 T T T T T T  7172 Fr3 A A A A A A  72 73 Fr3 D D D D D D  73 74 Fr3 T T T T T T  74 75Fr3 S S S S S S  75 76 Fr3 S T T T T T  76 77 Fr3 N D N N N D  77 78 Fr3T T T T T T  78 79 Fr3 A A A A A A  79 80 Fr3 Y Y Y Y Y Y  80 81 Fr3 L ML M L L  81 82 Fr3 Q E Q E Q Q  82 83 Fr3 L L L L L L  82A 84 Fr3 G S SS G G  82B 85 Fr3 S S S S S S  82C 86 Fr3 L L L L L L  83 87 Fr3 T R T TT T  84 88 Fr3 S S S S S S  85 89 Fr3 E E E E E E  86 90 Fr3 D D D D D D 87 91 Fr3 T T T T T T  88 92 Fr3 A A A A A A  89 93 Fr3 V V V V V V  9094 Fr3 Y Y Y Y Y Y  91 95 Fr3 F Y F Y F F  92 96 Fr3 C C A C C C C  9397 Fr3 S A E S S S S  94 98 CDR-H3 T T Y T T T T  95 99 CDR-H3 L F L L LL  96 CDR-H3  97 CDR-H3  98 CDR-H3  99 CDR-H3 100 CDR-H3 100A 100B 100C100D 100E 100F 100G 100H 100I 100J 100K 101 100 CDR-H3 D Q D D D D 102101 CDR-H3 F H F F F F 103 102 Fr4 W W W W W W 104 103 Fr4 G G G G G G105 104 Fr4 Q Q Q Q Q Q 106 105 Fr4 G G G G G G 107 106 Fr4 T T T T T T108 107 Fr4 T L L L L L 109 108 Fr4 L V V V V V 110 109 Fr4 T T T T T T111 110 Fr4 V V V V V V 112 111 Fr4 S S S S S S 113 112 Fr4 S S S S S SKabat h3D6VHv5 h3D6VHv1bA11B6G2 h3D6VHv1bA11B6H3 h3D6VHv1c h3D6VHv1dh3D6VHv1e residue (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID # NO:19) NO: 46) NO: 47) NO: 48) NO: 49) NO: 50)  1 E E E E E E  2 V V V V VV  3 Q Q Q Q Q Q  4 L L L L L L  5 V V V V V V  6 Q Q Q Q Q Q  7 S S S SS S  8 G G G G G G  9 A A A A A A  10 E E E E E D  11 V V V V V V  12 VV V K K V  13 K K K R R K  14 P P P P P P  15 G G G G G G  16 A A A A AA  17 T T T L L L  18 V V V V V V  19 K K K K K K  20 I I I I I I  21 SS S S S S  22 C C C C C C  23 K K K K K K  24 A A A A A A  25 S S S S SS  26 G G G G G G  27 F F F F Y F  28 T T T N T T  29 I I I F F I  30 KK K K T K  31 D D D D D D  32 Y Y Y Y Y Y  33 Y Y Y Y Y Y  34 L L L L LL  35 H H H H H H  35A  35B  36 W W W W W W  37 V V V V V V  38 R R R RR R  39 Q Q Q Q Q Q  40 R R R R R R  41 P P P P P P  42 G G G E E E  43Q K K Q Q Q  44 G G G G G G  45 L L L L L L  46 E E E E E E  47 W W W WW W  48 I I I M M I  49 G G G G G G  50 W W W W W W  51 I V I I V I  52D D D D D D  52A P P P P P P  52B  52C  53 E E E E E E  54 D D D N D N 55 G G G G G G  56 D D D D D D  57 T T T T T T  58 V V V V V V  59 Y YY Y Y Y  60 A A A D A A  61 P P P E E E  62 K K K K K K  63 F F F F F F 64 Q Q Q Q Q Q  65 G G G G G G  66 R R R R R R  67 A A A V V V  68 T TT T T T  69 I I I I I I  70 T T T T T T  71 A A A A A A  72 D D D D D D 73 T T T T T T  74 S S S S S S  75 T T T T T T  76 D D D N N N  77 T TT T T T  78 A A A A A A  79 Y Y Y Y Y Y  80 L L L L L L  81 Q E E Q Q E 82 L L L L L L  82A G G G G G G  82B S S S S S S  82C L L L L L L  83 TT T T T T  84 S S S S S S  85 E E E E E E  86 D D D D D D  87 T T T T TT  88 A A A A A A  89 V V V V V V  90 Y Y Y Y Y Y  91 F F F F F F  92 CC C C C C  93 S S S S S S  94 T T T T T T  95 L L L L L L  96  97  98 99 100 100A 100B 100C 100D 100E 100F 100G 100H 100I 100J 100K 101 D D DD D D 102 F F F F F F 103 W W W W W W 104 G G G G G G 105 Q Q Q Q Q Q106 G G G G G G 107 T T T T T T 108 L L L L L T 109 V V V V V L 110 T TT T T T 111 V V V V V V 112 S S S S S S 113 S S S S S S Kabat h3D6VHv1fh3D6VHv3 h3D6VHv3b h3D6VHv3c h3D6VHv4 h3D6VHv4b h3D6VHv4c residue (SEQID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID # NO: 51) NO: 52) NO:53) NO: 54) NO: 55) NO: 56) NO: 57)  1 E E E E E E E  2 V V V V V V V  3Q Q Q Q Q Q Q  4 L L L L L L L  5 V V V V V V V  6 Q Q Q Q Q Q Q  7 S SS S S S S  8 G G G G G G G  9 A A A A A A A  10 D E E E E E E  11 V V VV V V V  12 V K K K V V V  13 K K K K K K K  14 P P P P P P P  15 G G GG G G G  16 A A A A A A A  17 L T L L T L L  18 V V V V V V V  19 K K KK K K K  20 I I I I I I I  21 S S S S S S S  22 C C C C C C C  23 K K KK K K K  24 A V V V V V V  25 S S S S S S S  26 G G G G G G G  27 F F YY F Y Y  28 T N N T N N T  29 I I F F I F F  30 K K K T K K T  31 D D DD D D D  32 Y Y Y Y Y Y Y  33 Y Y Y Y Y Y Y  34 L L L L L L L  35 H H HH H H H  35A  35B  36 W W W W W W W  37 V V V V V V V  38 R R R R R R R 39 Q Q Q Q Q Q Q  40 R A A A R R R  41 P P P P P P P  42 G G G G G G G 43 Q K K K K K K  44 G G G G G G G  45 L L L L L L L  46 E E E E E E E 47 W W W W W W W  48 I M M M I M M  49 G G G G G G G  50 W W W W W W W 51 V I I V I I V  52 D D D D D D D  52A P P P P P P P  52B  52C  53 E EE E E E E  54 D N N D N N D  55 G G G G G G G  56 D D D D D D D  57 T TT T T T T  58 V V V V V V V  59 Y Y Y Y Y Y Y  60 A D D A D D A  61 E PE E P E E  62 K K K K K K K  63 F F F F F F F  64 Q Q Q Q Q Q Q  65 G GG G G G G  66 R R R R K R R  67 V V V V A V V  68 T T T T T T T  69 I II I I I I  70 T T T T T T T  71 A A A A A A A  72 D D D D D D D  73 T TT T T T T  74 S S S S S S S  75 T T T T T T T  76 D D N N N D D  77 T TT T T T T  78 A A A A A A A  79 Y Y Y Y Y Y Y  80 M M M M L L L  81 E EE E E E E  82 L L L L L L L  82A G S G G G G G  82B S S S S S S S  82C LL L L L L L  83 T R R R T T T  84 S S S S S S S  85 E E E E E E E  86 DD D D D D D  87 T T T T T T T  88 A A A A A A A  89 V V V V V V V  90 YY Y Y Y Y Y  91 F Y Y Y Y Y Y  92 C C C C C C C  93 S S S S S S S  94 TT T T T T T  95 L L L L L L L  96  97  98  99 100 100A 100B 100C 100D100E 100F 100G 100H 100I 100J 100K 101 D D D D D D D 102 Y F F F F F F103 W W W W W W W 104 G G G G G G G 105 Q Q Q Q Q Q Q 106 G G G G G G G107 T T T T T T T 108 T L L L L L L 109 L V V V V V V 110 T T T T T T T111 V V V V V V V 112 S S S S S S S 113 S S S S S S S

TABLE 4 Murine Hu VL Acceptor Fr Rabat Linear FR 3D6 VL Hu VL AcceptorFr Acc. # hu3D6VLv1 hu3D6VLv2 hu3D6VLv3 hu3D6VLv4 residue residue or(SEQ ID Acc. # IMGT#IGKV2- IMGT#IGKJ2*01 (SEQ ID (SEQ ID (SEQ ID (SEQ ID# # CDR NO: 11) 30*02 (SEQ ID NO: 27) (SEQ ID NO: 28) NO: 20) NO: 21)NO: 22) NO: 23)  1 1 Fr1 D D D D D D  2 2 Fr1 V V V V V I  3 3 Fr1 V V VV V V  4 4 Fr1 M M M M M M  5 5 Fr1 T T T T T T  6 6 Fr1 Q Q Q Q Q Q  77 Fr1 T S S S S T  8 8 Fr1 P P P P P P  9 9 Fr1 L L L L L L  10 10 Fr1 TS S S S S  11 11 Fr1 L L L L L L  12 12 Fr1 S P S P P S  13 13 Fr1 V V VV V V  14 14 Fr1 T T T T T T  15 15 Fr1 I L L L L I  16 16 Fr1 G G G G GG  17 17 Fr1 Q Q Q Q Q Q  18 18 Fr1 P P P P P P  19 19 Fr1 A A A A A A 20 20 Fr1 S S S S S S  21 21 Fr1 I I I I I I  22 22 Fr1 S S S S S S  2323 Fr1 C C C C C C  24 24 CDR-L1 K R K K K K  25 25 CDR-L1 S S S S S S 26 26 CDR-L1 S S S S S S  27 27 CDR-L1 Q Q Q Q Q Q  27A 28 CDR-L1 S S SS S S  27B 29 CDR-L1 L L L L L L  27C 30 CDR-L1 L V L L L L  27D 31CDR-L1 D H D D D D  27E 32 CDR-L1 S S S S S S  27F CDR-L1  28 33 CDR-L1D D D D D D  29 34 CDR-L1 G G G G G G  30 35 CDR-L1 K N K K K K  31 36CDR-L1 T T T T T T  32 37 CDR-L1 Y Y Y Y Y Y  33 38 CDR-L1 L L L L L L 34 39 CDR-L1 N N N N N N  35 40 Fr2 W W W W W W  36 41 Fr2 L F L L L L 37 42 Fr2 L Q L L L L  38 43 Fr2 Q Q Q Q Q Q  39 44 Fr2 R R R R R K  4045 Fr2 P P P P P P  41 46 Fr2 G G G G G G  42 47 Fr2 Q Q Q Q Q Q  43 48Fr2 S S S S S S  44 49 Fr2 P P P P P P  45 50 Fr2 K R K R R K  46 51 Fr2R R R R R R  47 52 Fr2 L L L L L L  48 53 Fr2 I I I I I I  49 54 Fr2 Y YY Y Y Y  50 55 CDR-L2 L K L L L L  51 56 CDR-L2 V V V V V V  52 57CDR-L2 S S S S S S  53 58 CDR-L2 K N K K K K  54 59 CDR-L2 L R L L L L 55 60 CDR-L2 D D D D D D  56 61 CDR-L2 S S S S S S  57 62 Fr3 G G G G GG  58 63 Fr3 V V V V V V  59 64 Fr3 P P P P P P  60 65 Fr3 D D D D S D 61 66 Fr3 R R R R R R  62 67 Fr3 F F F F F F  63 68 Fr3 T S S S S S  6469 Fr3 G G G G G G  65 70 Fr3 S S S S S S  66 71 Fr3 G G G G G G  67 72Fr3 S S S S S S  68 73 Fr3 G G G G G G  69 74 Fr3 T T T T T T  70 75 Fr3D D D D D D  71 76 Fr3 F F F F F F  72 77 Fr3 T T T T T T  73 78 Fr3 L LL L L L  74 79 Fr3 K K K K K K  75 80 Fr3 I I I I I I  76 81 Fr3 S S S SS S  77 82 Fr3 R R R R R R  78 83 Fr3 V V V V V V  79 84 Fr3 E E E E E E 80 85 Fr3 A A A A A A  81 86 Fr3 E E E E E E  82 87 Fr3 D D D D D D  8388 Fr3 L V V V V V  84 89 Fr3 G G G G G G  85 90 Fr3 V V V V V V  86 91Fr3 Y Y Y Y Y Y  87 92 Fr3 Y Y Y Y Y Y  88 93 Fr3 C C C C C C  89 94CDR-L3 W M W W W W  90 95 CDR-L3 Q Q Q Q Q Q  91 96 CDR-L3 G G G G G G 92 97 CDR-L3 T T T T T T  93 98 CDR-L3 H H H H H H  94 99 CDR-L3 F W FF F F  95 100 CDR-L3 P P P P P P  95A CDR-L3  95B CDR-L3  95C CDR-L3 95D CDR-L3  95E CDR-L3  95F CDR-L3  96 101 CDR-L3 Y Y Y Y Y Y  97 102CDR-L3 T T T T T T  98 103 Fr4 F F F F F F  99 104 Fr4 G G G G G G 100105 Fr4 G Q G G G G 101 106 Fr4 G G G G G G 102 107 Fr4 T T T T T T 103108 Fr4 K K K K K K 104 109 Fr4 L L L L L L 105 110 Fr4 E E E E E E 106111 Fr4 I I I I I I 106A 112 Fr4 K K K K K K 107 113 Fr4 R R R R R

TABLE 5 V_(H), V_(L) Backmutations and Other Mutations for Humanized 3D6Changes from Acceptor Framework (or CDR) Residues (based on V_(H) orV_(L) Variant V_(H) or V_(L) Exon Acceptor Sequence Kabat/ChothiaComposite CDRs) Hu3D6VHv NCBI accession code IMGT# IGHV1-69- H12, H13,H17, H24, H38, H42, (SEQ ID NO: 15) 2*01 (SEQ (ID NO: 25); NCBIaccession H43, H48, H66, H67, H76, H80, code IMGT# IGHJ1*01 (SEQ (ID NO:26) H81, H83, H91, H93 Hu3D6VHv2 NCBI accession code IMGT# IGHV1-69-H38, H42, H43, H76, H83, H93 (SEQ ID NO: 16) 2*01 (SEQ (ID NO: 25); NCBIaccession code IMGT# IGHJ1*01 (SEQ (ID NO: 26) Hu3D6VHv1b NCBI accessioncode IMGT# IGHV1-69- H12, H13, H17, H24, H38, H40, (SEQ ID NO: 17) 2*01(SEQ (ID NO: 25); NCBI accession H42, H43, H48, H66, H67, H76, codeIMGT# IGHJ1*01 (SEQ (ID NO: 26) H80, H81, H82A, H83, H91, H93Hu3D6VHvlbA11 NCBI accession code IMGT# IGHV1-69- H12, H24, H38, H40,H43, H48, (SEQ ID NO: 18) 2*01 (SEQ (ID NO: 25); NCBI accession H67,H80, H81, H82A, H83, H91, code IMGT# IGHJ1*01 (SEQ (ID NO: 26) H93Hu3D6VHv5 NCBI accession code IMGT# IGHV1-69- H12, H24, H28, H38, H40,H43, (SEQ ID NO: 19) 2*01 (SEQ (ID NO: 25); NCBI accession H48, H54,H60, H67, H80, H81, code IMGT# IGHJ1*01 (SEQ (ID NO: 26) H82A, H83, H91,H93 Hu 3D6VHv1bA11B6G2 NCBI accession code IMGT# IGHV1-69- H12, H24,H28, H38, H40, H48, (SEQ ID NO: 46) 2*01 (SEQ (ID NO: 25); NCBIaccession H51, H54, H60, H67, H80, H82A, code IMGT# IGHJ1*01 (SEQ (IDNO: 26) H83, H91, H93 Hu 3D6VHv1bA11B6H3 NCBI accession code IMGT#IGHV1-69- H12, H24, H28, H38, H40, H48, (SEQ ID NO: 47) 2*01 (SEQ (IDNO: 25); NCBI accession H54, H60, H67, H80, H82A, H83, code IMGT#IGHJ1*01 (SEQ (ID NO: 26) H91, H93 Hu3D6VHv1c NCBI accession code IMGT#IGHV1-69- H13, H17, H24, H29, H38, H40, (SEQ ID NO: 48) 2*01 (SEQ (IDNO: 25); NCBI accession H42, H43, H54, H61, H76, H80, code IMGT#IGHJ1*01 (SEQ (ID NO: 26) H81, H82A, H83, H91, H93 Hu3D6VHv1d NCBIaccession code IMGT# IGHV1-69- H13, H17, H24, H27, H28, H29, (SEQ ID NO:49) 2*01 (SEQ (ID NO: 25); NCBI accession H30, H38, H40, H42, H43, H51,code IMGT# IGHJ1*01 (SEQ (ID NO: 26) H54, H60, H61, H76, H80, H81, H82A,H83, H91, H93 Hu3D6VHv1e NCBI accession code IMGT# IGHV1-69- H10, H12,H17, H24, H28, H38, (SEQ ID NO: 50) 2*01 (SEQ (ID NO: 25); NCBIaccession H40, H42, H43, H48, H54, H60, code IMGT# IGHJ1*01 (SEQ (ID NO:26) H61, H76, H80, H82A, H83, H91, H93, H108, H109 Hu3D6VHv1f NCBIaccession code IMGT# IGHV1-69- H10, H12, H17, H24, H28, H38, (SEQ ID NO:51) 2*01 (SEQ (ID NO: 25); NCBI accession H40, H43, H48, H51, H54, H60,code IMGT# IGHJ1*01 (SEQ (ID NO: 26) H61, H82A, H83, H91, H93, H102,H108, H109 Hu3D6VHv3 NCBI accession code IMGT# IGHV1-69- H38, H93 (SEQID NO: 52) 2*01 (SEQ (ID NO: 25); NCBI accession code IMGT# IGHJ1*01(SEQ (ID NO: 26) Hu3D6VHv3b NCBI accession code IMGT# IGHV1-69- H17,H27, H29, H38, H61, H76, (SEQ ID NO: 53) 2*01 (SEQ (ID NO: 25); NCBIaccession H82A, H93 code IMGT# IGHJ1*01 (SEQ (ID NO: 26) Hu3D6VHv3c NCBIaccession code IMGT# IGHV1-69- H17, H27, H28, H29, H30, H38, (SEQ ID NO:54) 2*01 (SEQ (ID NO: 25); NCBI accession H51, H54, H60, H61, H76, H82A,code IMGT# IGHJ1*01 (SEQ (ID NO: 26) H93 Hu3D6VHv4 NCBI accession codeIMGT# IGHV1-69- H12, H38, H40, H48, H66, H67, (SEQ ID NO: 55) 2*01 (SEQ(ID NO: 25); NCBI accession H76, H80, H82A, H83, H93 code IMGT# IGHJ1*01(SEQ (ID NO: 26) Hu3D6VHv4b NCBI accession code IMGT# IGHV1-69- H12,Hl7, H27, H29, H38, H40, (SEQ ID NO: 56) 2*01 (SEQ (ID NO: 25); NCBIaccession H61, H80, H82A, H83, H93 code IMGT# IGHJ1*01 (SEQ (ID NO: 26)Hu3D6VHv4c NCBI accession code IMGT# IGHV1-69- H12, H17, H27, H28, H29,H30, (SEQ ID NO: 57) 2*01 (SEQ (ID NO: 25); NCBI accession H38, H40,H51, H54, H60, H61, code IMGT# IGHJ1*01 (SEQ (ID NO: 26) H80, H82A, H83,H93 Hu3D6VLv1 NCBI accession code IMGT#IGKV2- L12, L36, L37, L45, L100(SEQ ID NO: 20) 30*02 (SEQ (ID NO: 27); NCBI accession codeIMGT#IGKJ2*01 (SEQ (ID NO: 28) Hu3D6VLv2 NCBI accession code IMGT#IGKV2-L36, L37, L100 (SEQIDN0:21) 30*02 (SEQ (ID NO: 27); NCBI accession codeIMGT#IGKJ2*01 (SEQ (ID NO: 28) Hu3D6VLv3 NCBI accession code IMGT#IGKV2-L36, L37, L60, L100 (SEQ ID NO: 22) 30*02 (SEQ (ID NO: 27); NCBIaccession code IMGT#IGKJ2*01 (SEQ (ID NO: 28) Hu3D6VLv4 NCBI accessioncode IMGT#IGKV2- L2, L7, L12, L15, L36, L37, L45, (SEQ ID NO: 23) 30*02(SEQ (ID NO: 27); NCBI accession L100 code IMGT#IGKJ2*01 (SEQ (ID NO:28)

TABLE 6 Kabat Numbering of Framework (or CDR) Residues (based onKabat/Chothia Composite CDRs) for Backmutations and Other Mutations inHeavy Chains of Humanized 3D6 Antibodies (Heavy (Heavy Chain)IMGT#Chain) IMGT# Mouse Hu3D6 Hu3D6 Hu3D6 Hu3D6 Hu3D6 Residue IGHV1-69-2*01IGHJ1*01 3D6 VHv1 VHv2 VHv1b VHv1bA11 VHv5 H10 E D E E E E E H12 K V V KV V V H13 K R R K R K K H17 T L L S L T T H24 V A A V A A A H27 Y F F FF F F H28 T N N N N N T H29 F I I I I I I H30 T K K K K K K H38 Q R R RR R R H40 A R A A R R R H42 G E E E E G G H43 K Q Q Q Q Q Q H48 M I I MI I I H51 V I I I I I I H54 D N N N N N D H60 A D D D D D A H61 E P P PP P P H66 R K K R K R R H67 V A A V A A A H76 D N N N N D D H80 M L L ML L L H81 E Q Q E Q Q Q H82A S G S S G G G H83 R T T T T T T H91 Y F F YF F F H93 A E S S S S S S H102 H F F F F F F H108 L T L L L L L H109 V LV V V V V Hu Hu Hu 3D6 3D6 3D6 Residue Hu3D6VHv1bA11B6G2Hu3D6VHv1bA11B6H3 VHv1c VHv1d VHv1e H10 E E E E D H12 V V K K V H13 K KR R K H17 T T L L L H24 A A A A A H27 F F F Y F H28 T T N T T H29 I I FF I H30 K K K T K H38 R R R R R H40 R R R R R H42 G G E E E H43 K K Q QQ H48 I I M M I H51 V I I V I H54 D D N D N H60 A A D A A H61 P P E E EH66 R R R R R H67 A A V V V H76 D D N N N H80 L L L L L H81 E E Q Q EH82A G G G G G H83 T T T T T H91 F F F F F H93 S S S S S H102 F F F F FH108 L L L L T H109 V V V V L Hu Hu Hu Hu Hu Hu Hu 3D6 3D6 3D6 3D6 3D63D6 3D6 Residue VHv1f VHv3 VHv3b VHv3c VHv4 VHv4b VHv4c H10 D E E E E EE H12 V K K K V V V H13 K K K K K K K H17 L T L L T L L H24 A V V V V VV H27 F F Y Y F Y Y H28 T N N T N N T H29 I I F F I F F H30 K K K T K KT H38 R R R R R R R H40 R A A A R R R H42 G G G G G G G H43 Q K K K K KK H48 I M M M I M M H51 V I I V I I V H54 D N N D N N D H60 A D D A D DA H61 E P E E P E E H66 R R R R K R R H67 V V V V A V V H76 D D N N N DD H80 M M M M L L L H81 E E E E E E E H82A G S G G G G G H83 T R R R T TT H91 F Y Y Y Y Y Y H93 S S S S S S S H102 Y F F F F F F H108 T L L L LL L H109 L V V V V V V

TABLE 7 Table 7: Kabat Numbering of Framework Residues (based onKabat/Chothia Composite CDRs) for Backmutations and Other Mutations inLight Chains of Humanized 3D6 Antibodies (Light Chain) (LightIMGT#IGKV2- Chain) Mouse Residue 30*02 IMGT#IGKJ2*01 3D6 Hu3D6VLv1Hu3D6VLv2 Hu3D6VLv3 Hu3D6VLv4 L2 V V V V V I L7 S T S S S T L12 P S S PP S L15 L I L L L I L36 F L L L L L L37 Q L L L L L L45 R K K R R K L60D D D D S D L100 Q G G G G G

TABLE 8 Percentage Humanness of Heavy and Light Chains of Humanized 3D6Antibodies V_(H) or V_(L) Variant % Humanness Hu3D6VHv1 (SEQ ID NO: 15)70.40%  Hu3D6VHv2 (SEQ ID NO: 16) 79.60%  Hu3D6VHv1b (SEQ ID NO: 17)69.40%  Hu3D6VHv1bA11 (SEQ ID NO: 18) 74.50%  Hu3D6VHv5 (SEQ ID NO: 19)77.60%  Hu3D6VHv1bA11B6G2 (SEQ ID NO: 46) 80.6% Hu3D6VHv1bA11B6H3 (SEQID NO: 47) 79.6% Hu3D6VHv1c (SEQ ID NO: 48) 75.5% Hu3D6VHv1d (SEQ ID NO:49) 81.6% Hu3D6VHv1e (SEQ ID NO: 50) 75.5% Hu3D6VHv1f (SEQ ID NO: 51)80.6% Hu3D6VHv3 (SEQ ID NO: 52) 85.7% Hu3D6VHv3b (SEQ ID NO: 53) 85.7%Hu3D6VHv3c (SEQ ID NO: 54) 90.8% Hu3D6VHv4 (SEQ ID NO: 55) 76.5%Hu3D6VHv4b(SEQ ID NO: 56) 82.7% Hu3D6VHv4c (SEQ ID NO: 57) 87.8%Hu3D6VLv1 (SEQ ID NO: 20)  87% Hu3D6VLv2 (SEQ ID NO: 21)  89% Hu3D6VLv3(SEQ ID NO: 22)  88% Hu3D6VLv4 (SEQ ID NO: 23)  87%

Positions at which canonical, vernier, or interface residues differbetween mouse and human acceptor sequences are candidates forsubstitution. Examples of canonical/CDR interacting residues includeKabat residues H24, H27, H29, H34, H38, and H67 in Tables 3 and 4.Examples of interface/packing (VH+VL) residues include Kabat residuesH40, H43, H48, H91, H93, L36, L98, and L100 in Tables 3 and 4.

The rationales for selection of the positions indicated in Table 4 inthe light chain variable region as candidates for substitution are asfollows.

Q100G is a mutation of a residue that contacts an interface residue.F36L and M89W are mutations of an interface residue. V2I is afrequency/germ-line aligning mutation. S7T is a mutation of a residuethat is similar to S and may have similar immunogenicity. P12S is amutation to try to increase contact with E105. L15I is a mutation to aresidue that is similar to L. R45K is a mutation to a residue that issimilar to R. D60S is a mutation to S as D is a predicted proteasecleavage site.

The rationales for selection of the positions indicated in Table 3 inthe heavy chain variable region as candidates for substitution are asfollows.

V24A is a mutation of a HCDR1 canonical residue. V67A is a mutation of aresidue that contacts HCDR2. Q38R is a mutation of a canonical/CDRinteracting residue. A40R, K43Q, and M48I are mutations of residues thatcontact interface residues. Y91F and A93S are mutations of interfaceresidues. K12V, M80L, and E81Q are frequency based back-mutations orgerm-line aligning mutations. S82aG is a mutation to test for increasedbinding.

S at Kabat residue H17 in hu3D6VHv2 was acquired from human VH acceptorAcc. #BAC01986.1.

V at Kabat residue H20 in hu3D6VHv1 and hu3D6VHv2 were acquired fromhuman VH acceptor Acc. #BAC01986.1. Y at Kabat residue H102 inhu3D6VHv1f was acquired from the Mus VH structure template (PDB #1CR9_H;SEQ ID NO:70) and from mouse anti-tau monoclonal antibody 6A10.

Humanized sequences are generated using a two-stage PCR protocol thatallows introduction of multiple mutations, deletions, and insertionsusing QuikChange site-directed mutagenesis [Wang, W. and Malcolm, B. A.(1999) BioTechniques 26:680-682).

The designs based on these human frameworks were:

VARIABLE KAPPA hu3D6VLv1 (SEQ ID NO: 20):DVVMTQSPLSLSVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIKRhu3D6VLv2 (SEQ ID NO: 21):DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIKRhu3D6VLv3 (SEQ ID NO: 22):DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVSKLDSGVPSRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIKRhu3D6VLv4 (SEQ ID NO: 24):DIVMTQTPLSLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQKPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIKR VARIABLE HEAVYhu3D6VHv1 (SEQ ID NO: 15):EVQLVQSGAEVVRPGALVKVSCKASGFNIKDYYLHWVRQAPEQGLEWIGWIDPENGDTVYDPKFQGKATITADTSTNTAYLQLSSLTSEDTAVYFCSTLDFWGQGTLVTVSShu3D6VHv2 (SEQ ID NO: 16):EVQLVQSGAEVKKPGASVKVSCKVSGFNIKDYYLHWVRQAPEQGLEWMGWIDPENGDTVYDPKFQGRVTITADTSTNTAYMELSSLTSEDTAVYYCSTLDFWGQGTLVTVSShu3D6VHv1b (SEQ ID NO: 17):EVQLVQSGAEVVRPGALVKISCKASGFNIKDYYLHWVRQRPEQGLEWIGWIDPENGDTVYDPKFQGKATITADTSTNTAYLQLGSLTSEDTAVYFCSTLDFWGQGTLVTVSShu3D6VHv1bA11 (SEQ ID NO: 18):EVQLVQSGAEVVKPGATVKISCKASGFNIKDYYLHWVRQRPGQGLEWIGWIDPENGDTVYDPKFQGRATITADTSTDTAYLQLGSLTSEDTAVYFCSTLDFWGQGTLVTVSShu3D6VHv5 (SEQ ID NO: 19):EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGQGLEWIGWIDPEDGDTVYAPKFQGRATITADTSTDTAYLQLGSLTSEDTAVYFCSTLDFWGQGTLVTVSShu3D6VHv1bA11B6G2 (SEQ ID NO: 46):EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWVDPEDGDTVYAPKFQGRATITADTSTDTAYLELGSLTSEDTAVYFCSTLDFWGQGTLVTVSShu3D6VHv1bA11B6H3 (SEQ ID NO: 47):EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPEDGDTVYAPKFQGRATITADTSTDTAYLELGSLTSEDTAVYFCSTLDFWGQGTLVTVSShu3D6VHv1c (SEQ ID NO: 48):EVQLVQSGAEVKRPGALVKISCKASGFNFKDYYLHWVRQRPEQGLEWMGWIDPENGDTVYDEKFQGRVTITADTSTNTAYLQLGSLTSEDTAVYFCSTLDFWGQGTLVTVSShu3D6VHv1d (SEQ ID NO: 49):EVQLVQSGAEVKRPGALVKISCKASGYTFTDYYLHWVRQRPEQGLEWMGWVDPEDGDTVYAEKFQGRVTITADTSTNTAYLQLGSLTSEDTAVYFCSTLDFWGQGTLVTVSShu3D6VHv1e (SEQ ID NO: 50:EVQLVQSGADVvkPGALVKISCKASGFTIKDYYLHWVRQRPEQGLEWIGWIDPENGDTVYAEKFQGRVTITADTSTNTAYLeLGSLTSEDTAVYFCSTLDFWGQGTTLTVSShu3D6VHv1f (SEQ ID NO: 51):EVQLVQSGADVVKPGALVKISCKASGFTIKDYYLHWVRQRPGQGLEWIGWVDPEDGDTVYAEKFQGRVTITADTSTDTAYMELGSLTSEDTAVYFCSTLDYWGQGTTLTVSShu3D6VHv3 (SEQ ID NO: 52):EVQLVQSGAEVKKPGATVKISCKVSGFNIKDYYLHWVRQAPGKGLEWMGWIDPENGDTVYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCSTLDFWGQGTLVTVSShu3D6VHv3b (SEQ ID NO: 53):EVQLVQSGAEVKKPGALVKISCKVSGYNFKDYYLHWVRQAPGKGLEWMGWIDPENGDTVYDEKFQGRVTITADTSTNTAYMELGSLRSEDTAVYYCSTLDFWGQGTLVTVSShu3D6VHv3c (SEQ ID NO: 54):EVQLVQSGAEVKKPGALVKISCKVSGYTFTDYYLHWVRQAPGKGLEWMGWVDPEDGDTVYAEKFQGRVTITADTSTNTAYMELGSLRSEDTAVYYCSTLDFWGQGTLVTVSShu3D6VHv4 (SEQ ID NO: 55):EVQLVQSGAEVVKPGATVKISCKVSGFNIKDYYLHWVRQRPGKGLEWIGWIDPENGDTVYDPKFQGKATITADTSTNTAYLELGSLTSEDTAVYYCSTLDFWGQGTLVTVSShu3D6VHv4b (SEQ ID NO: 56):EVQLVQSGAEVVKPGALVKISCKVSGYNFKDYYLHWVRQRPGKGLEWMGWIDPENGDTVYDEKFQGRVTITADTSTDTAYLELGSLTSEDTAVYYCSTLDFWGQGTLVTVSShu3D6VHv4c (SEQ ID NO: 57):EVQLVQSGAEVVKPGALVKISCKVSGYTFTDYYLHWVRQRPGKGLEWMGWVDPEDGDTVYAEKFQGRVTITADTSTDTAYLELGSLTSEDTAVYYCSTLDFWGQGTLVTVSS

Example 4. Mouse Monoclonal Antibodies Bind Tau in ELISA Assays

Methods: Indirect ELISA: 96-well polystyrene plates were coated withcapture antibodies anti-6×His (FIG. 5A) or polyclonal anti-tau (Dako#A0024, FIG. 5B) suspended in 1×PBS for 2 hr at RT or 16 hr at 4° C.Coating was removed, and plates were blocked for 1 hr with 1% BSA in1×PBS, followed by incubation with human recombinant tau, either with(FIG. 5A) or without (FIG. 5B) a polyhistidine tag at the N-terminus ofthe protein. After washing, plates were incubated with indicatedantibodies, washed, and incubated with HRP-conjugated goat anti-mousesecondary antibody. Plates were developed with TMB, and A₄₅₀ wasmeasured with a plate reader.

Sandwich ELISA: 96-well polystyrene plates were coated with anti-mouseantibodies in 1×PBS for 2 hr at RT or 16 hr at 4° C. Coating wasremoved, and plates were blocked for 1 hr with 1% BSA in 1×PBS. Theplate was next incubated with the Indicated antibodies at identicalconcentrations, diluted in 0.1% BSA in 1×PBS. Plates were successivelytreated with human tau, polyclonal rabbit anti-tau (Dako #A0024), andHRP-conjugated goat anti-rabbit antibody, all diluted in 0.1% BSA in PBSwith washes occurring between each step. Streptavidin-HRP was added,plates were developed with TMB, and A450 was measured with a platereader. See FIG. 5C.

Results: A panel of hybridoma-produced antibodies were assayed forbinding to tau via a number of different ELISA formats. Detection of tauwas confirmed using an indirect format, using tau protein immobilized byits N-terminally fused polyhistidine tag (FIG. 5A). Binding to thenative, untagged protein was also confirmed (FIG. 5B). To assess thesolution affinity of the various antibodies, a sandwich ELISA format wasused in which tested hybridoma antibodies were used as capture reagents(FIG. 5C).

Example 5. Affinity of Mouse Monoclonal Antibodies to Tau

Methods: SPR analysis was performed using a Biacore T200 to determinethe binding kinetics of murine antibodies to recombinant human tau. Toprepare a sensor surface, anti-mouse antibody (GE Life Sciences) wasimmobilized on sensor chip CMS via amine coupling, and antibody wascaptured at a level to ensure maximum binding of 50 RU. Variousconcentrations of recombinant tau ranging from 10-0.14 nM were passedover the captured ligand at a flow rate of 50 μL/min in running buffer(HBS+0.05% P-20, 1 mg/mL BSA), for 180 sec association and 900 secdissociation. Data were double-referenced to both an irrelevant sensornot containing antibody ligand, and 0 nM analyte concentration toaccount for the dissociation of ligand from the capture moiety. Data wasthen analyzed using a global 1:1 fit.

Results: Multiple murine antibodies were selected based on theirperformance in a battery of ELISA assays, and their binding affinitieswere assessed via SPR. Antibodies were tested in parallel sets, andtheir binding association and dissociation rates were compared to selectthe highest binder to recombinant human tau. The highest bindingaffinity was observed with antibody clone 3D6. Binding affinities areshown in FIG. 6 .

Example 6. Mouse Monoclonal Antibodies Prevent Binding of Human Tau tothe Surface of Immortalized Neuronal Cells

Methods: Inhibition of Tau Binding to B103 Neuroblastoma Cells withanti-Tau Monoclonal Antibodies

1. Resuspend B103 cells in PBS at 5×10⁵ cells/mL. Plate 50 μL of cellsuspension per well in a MSD High Bind plate. This results in 25Kcells/well. Cover the plate and allow cells to attach at 37° C., 5% CO₂,for 2 hrs.

2. Following cell attachment, remove PBS from wells by inverting plateand gently tapping to remove excess buffer. Add 504, of 3% MSD Blocker Ain PBS or other suitable blocking buffer to each well and incubate plateat RT for 1 hr without shaking.

3. During the plate blocking step co-incubate Tau and anti-Tauantibodies as follows:

a. Start with anti-Tau antibody at 2 mg/mL and serial dilute in PBS,1:2, for 7 additional dilutions.

b. Dilute Tau to 20 nM in PBS. The Tau concentration will be constant ineach well.

c. Mix the Tau and anti-Tau antibody, 1:1, for a final Tau concentrationof 10 nM and a starting concentration of anti-Tau of 1 mg/mL.

d. Incubate the mixture for approximately 1 hr at RT with shaking (600rpm).

4. After plate blocking, step 2, remove blocking buffer from wells byinverting plate and gently tapping and wash plate 2× with PBS using amultichannel pipette. Ensure excess buffer is completely removed. Coolthe plated cells to 4° C. prior to adding the Tau: anti-Tau complexes.5. Add 504, of cooled complex, step 3, to the plated cells and incubateon ice for 30 minutes.6. Wash plate 2× with chilled PBS as previously described.7. Add 504, per well of the 16B5.SULFO-TAG for detection of cell surfacebound Tau. Incubate for 30 minutes on ice.8. Wash plate 2× with chilled PBS again as previously described.9. Add 150 μL per well of 1× Read Buffer T Without Surfactant (dilutedin H₂O) and read immediately on the MSD SECTOR™ 600 instrument. Avoidintroducing bubbles when adding read buffer.10. Report the MSD signals vs. concentration of anti-Tau.

Antibodies tested were anti-tau antibodies 3D6, 16G7, 3H9, 4C5, and 5G8,and isotype control.

Results:

Decreasing SulfoTag anti-tau signal occurring with increasing testantibody indicates functional blocking of the binding of tau to neuronalcell surfaces. No blocking was observed with isotype control, 16G7, or3H9. Increasing amounts of functional blocking activity were observedwith 4C5, 5G8, and 3D6. 3D6 demonstrated the deepest blocking activityof the antibodies tested. See FIG. 7 .

Example 7. Disaggregation Activity

Methods: Aggregation of recombinant tau—Purified recombinant tau with anN-terminal 6×His tag was combined with equimolar amounts oflow-molecular weight heparin in 1×PBS (pH 7.4), and incubated at 37° C.for 96 hr on a nutator. Aggregation of the sample was confirmed bybinding to Thioflavin T.

Incubation with antibodies—Antibodies were incubated with aggregated,recombinant tau at the indicated molar ratios incubated at 37° C. for 96hr without rotation or nutation. At the end of the experiment,aggregation was measured by incubating samples with 25 mM Thioflavin T,and measuring emitted fluorescence (450/482 ex/em). Signals werebackground subtracted to buffer samples.

Results: As shown in FIG. 8 , 3D6 preferentially disassembles intact taufibrils. Varying molar ratios of 3D6 (triangles), isotype control(circles) and 16G7 (squares) were incubated with amyloid-containing taufibrils for 96 hours. At the end of this period, the extent ofaggregation was assessed by binding to Thioflavin T. 3D6 preferentiallydecreases the Thioflavin T signal present in the sample, compared toboth an isotype control antibody as well as to 16G7, an anti-tauantibody that binds to a different region of tau.

Example 8. 3D6 and 5G8 Immunocapture Tau from Human Disease Tissue

Methods: High-salt soluble protein fractions were prepared to 1 mg/ml.For each immunoprecipitation, 200 μg of sample was used. 10 μg of theindicated antibody (either an isotype control, 3D6, or anti-tau antibody5G8) was added to the high-salt sample preparations, and incubated for 2hr. Protein G magnetic beads were then added to the mixtures, andincubated for a further hour to capture antibody/antigen complexes.Samples were thoroughly washed with 1×PBS, and beads were boiled inreducing/denaturing sample buffer to release captured proteins.Resulting samples were resolved by SDS-PAGE and Western blotting wasperformed using a polyclonal anti-tau antibody (Dako, #A0024).

Results: As shown in FIG. 9 , 3D6 and 5G8 immunoprecipitated tau fromAlzheimer disease tissue. High-salt soluble fractions wereimmunoprecipitated with the indicated antibody, and detected with apolyclonal anti-tau antibody directed towards a separate region of thetau molecule from the binding sites for 3D6 and tau antibody A. 3D6robustly captured tau from this fraction. The input (high-salt solublesample) is shown at right.

Example 9. Immunohistochemistry Immunoreactivity of 3D6

Frontotemporal cortices were obtained from patients withoutneurodegenerative disease or with Alzheimer disease, which was confirmedupon post-mortem assessment. Immunohistochemistry was performed onlightly acetone-fixed, 10 um slide-mounted cryosections. All stainingsteps were performed using a Leica BOND Rx autostainer, using Leicaconsumables. Either murine or a human form of 3D6 was incubated withtissue sections followed by addition of species-appropriate secondaryantibodies conjugated to an HRP polymer. To prevent non-specific bindingof endogenous immunoglobulin when using humanized antibodies on humantissue, the antibodies were non-covalently labeled with abiotin-conjugated anti-human monovalent Fab fragment in vitro beforeincubation on tissue. Tissue labeled with the primary antibody-biotinFab fragment complex was further amplified using an avidin-biotinamplification system (Vector Laboratories, Burlingame, Calif.). Thestaining was visualized with a DAB chromogen, which produced a browndeposit. Negative control consisted of performing the entireimmunohistochemical procedure on adjacent sections with an IgG isotypecontrol antibody.

Antibodies tested were murine CD6, chimeric 3D6 (which contained VH andVL from the murine antibody with human constant regions, heavy chain SEQID NO:72 and light chain SEQ ID NO:73), and humanized varianthu3D6VHv5/hu3D6VLv2.

Staining performed with murine, chimeric, and humanized forms of 3D6were qualitatively compared and assessed for the strength and intensityof staining, as well as localization of immunoreactivity. Intensity ofstaining was similar for chimeric and humanized forms of 3D6, anddisplayed similar localization patterns compared with the murine form ofthe antibody. Tau was detected in neurofibrillary tangles, fibrils,neuropil threads, and in degenerating axons. There was also notablesomal staining detected.

Example 10. Affinity of Humanized Variants Towards Tau

Methods; Indirect ELISA 96-well polystyrene plates were coated withhuman recombinant tau suspended in 1×PBS for 2 h at RT or 16 h at 4° C.Coating was removed, and plates were blocked for 1 h with 1% BSA in1×PBS. Indicated antibodies at 1 μg/mL in 0.1% BSA in 1×PBS were addedto plates for 1 hour followed by washing, and HRP-conjugated goatanti-human antibody was added. Plates were developed with TMB, and A450was measured with a plate reader.

Sandwich ELISA 96-well polystyrene plates were coated with anti-humanantibodies in 1×PBS for 2 hr at RT or 16 hr at 4° C. Coating wasremoved, and plates were blocked for 1 hr with 1% BSA in 1×PBS.Indicated antibodies at varying concentrations as indicated, diluted in0.1% BSA in 1×PBS were added to plates for 1 hour followed by washing,and biotinylated recombinant human tau diluted in 0.1% BSA in 1×PBS wasadded. After washing, Streptavidin-HRP was added, plates were developedwith TMB, and A450 was measured with a plate reader.

SPR analysis was performed using a Biacore T200 to determine the bindingkinetics of h3D6-VHv5-L2 to recombinant human tau. To prepare a sensorsurface, anti-human antibody (GE Life Sciences) was immobilized onsensor chip CMS via amine coupling, and hu3D6VHv5/hu3D6VLv2 was capturedat a level to ensure maximum binding of 50 RU. Various concentrations ofrecombinant tau ranging from 10-0.14 nM were passed over the capturedligand at a flow rate of 50 μL/min in running buffer (HBS+0.05% P-20, 1mg/mL BSA), for 180 s association and 900 s dissociation. Data weredouble-referenced to both an irrelevant sensor not containing antibodyligand, and 0 nM analyte concentration to account for the dissociationof ligand from the capture moiety. Data was then analyzed using a global1:1 fit.

Antibodies tested were chimeric 3D6 (comprising murine VH and VL regionswith human constant regions; SEQ ID NO:72 (heavy chain) and SEQ ID NO:73(light chain), and humanized variants (hu3D6VHv1b/hu3D6VLv2,hu3D6VHv1c/hu3D6VLv2, hu3D6VHv1d/hu3D6VLv2, hu3D6VHv1e/hu3D6VLv2,hu3D6VHv1f/hu3D6VLv2, hu3D6VHv2/hu3D6VLv2, hu3D6VHv3/hu3D6VLv2,hu3D6VHv3b/hu3D6VLv2, hu3D6VHv3c/hu3D6VLv2, hu3D6VHv4/hu3D6VLv2,hu3D6VHv4b/hu3D6VLv2, hu3D6VHv4c/hu3D6VLv2, hu3D6VHv5/hu3D6VLv2,hu3D6VHv1bB6A11/hu3D6VLv2, hu3D6VHv1bB6A11B6G2/hu3D6VLv2,hu3D6VHv1bB6A11B6H2/hu3D6VLv2).

FIG. 10A: Multiple candidate sequences from the humanization of 3D6 weretested via ELISA to compare binding to recombinant human tau. Absorbancevalues were compared and normalized to the value for chimeric-3D6, whichcontained VH and VL from the murine antibody. Multiple candidatesequences had similar binding when compared to chimeric antibody, withhu3D6VHv5/hu3D6VLv2 displaying the highest reactivity.

FIG. 10B: Binding of the hu3D6VHv5/hu3D6VLv2 was compared withchimeric-3D6 via a concentration curve in a sandwich ELISA, withchimeric-3D6 and hu3D6VHv5/hu3D6VLv2 used as tau capture reagents. Bothantibodies displayed similar binding curves, indicating similaraffinities.

FIG. 10C: Affinity of hu3D6VHv5/hu3D6VLv2 was determined by SPRanalysis. Varying concentrations of recombinant tau were flowed overimmobilized antibody, and resulting sensorgrams were analyzed using aglobal 1:1 fit. The kinetic affinity, association, and dissociationrates are shown here, and compare very favorably with murine 3D6 values.

Listing of Sequences P10636-8 (SEQ ID NO: 1)MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL P10636-7 (SEQ ID NO: 2)MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGLP10636-6 (4RON human tau) (SEQ ID NO: 3)MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL P10636-5 (SEQ ID NO: 4)MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGLP10636-4 (SEQ ID NO: 5)MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL P10636-2 (SEQ ID NO: 6)MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGLSEQ ID NO: 7; Murine 3D6 VH amino acid sequence:EVQLQQSGADLVRPGALVKLSCKASGFNIKDYYLHWVRQRPEQGLEWIGWIDPENGDTVYDPKFQGKATITADTSSNTAYLQLGSLTSEDTAVYFCSTLDFWGQGTTLTVSSSEQ ID NO: 8; Kabat/Chothia HCDR1: GFNIKDYYLH SEQ ID NO: 9; Kabat HCDR2:WIDPENGDTVYDPKFQG SEQ ID NO: 10; Kabat HCDR3: LDFSEQ ID NO: 11; Murine 3D6 VL amino acid sequence:DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKLEIKRSEQ ID NO: 12; Murine Kabat LCDR1: KSSQSLLDSDGKTYLNSEQ ID NO: 13; Murine Kabat LCDR2: LVSKLDSSEQ ID NO: 14; Murine Kabat LCDR3: WQGTHFPYT SEQ ID NO: 15; hu3D6VHv1:EVQLVQSGAEVVRPGALVKVSCKASGFNIKDYYLHWVRQAPEQGLEWIGWIDPENGDTVYDPKFQGKATITADTSTNTAYLQLSSLTSEDTAVYFCSTLDFWGQGTLVTVSSSEQ ID NO: 16; hu3D6VHv2:EVQLVQSGAEVKKPGASVKVSCKVSGFNIKDYYLHWVRQAPEQGLEWMGWIDPENGDTVYDPKFQGRVTITADTSTNTAYMELSSLTSEDTAVYYCSTLDFWGQGTLVTVSSSEQ ID NO: 17; hu3D6VHv1b:EVQLVQSGAEVVRPGALVKISCKASGFNIKDYYLHWVRQRPEQGLEWIGWIDPENGDTVYDPKFQGKATITADTSTNTAYLQLGSLTSEDTAVYFCSTLDFWGQGTLVTVSSSEQ ID NO: 18; hu3D6VHv1bA11:EVQLVQSGAEVVKPGATVKISCKASGFNIKDYYLHWVRQRPGQGLEWIGWIDPENGDTVYDPKFQGRATITADTSTDTAYLQLGSLTSEDTAVYFCSTLDFWGQGTLVTVSSSEQ ID NO: 19; hu3D6VHv5:EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGQGLEWIGWIDPEDGDTVYAPKFQGRATITADTSTDTAYLQLGSLTSEDTAVYFCSTLDFWGQGTLVTVSSSEQ ID NO: 20; hu3D6VLv1:DVVMTQSPLSLSVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIKRSEQ ID NO: 21; hu3D6VLv2:DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIKRSEQ ID NO: 22; hu3D6VLv3:DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVSKLDSGVPSRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIKRSEQ ID NO: 23; hu3D6VLv4:DIVMTQTPLSLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQKPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIKRSEQ ID NO: 24; heavy chain variable acceptor Acc.# BAC01986.1QVQLQQSGAEVKKPGSSVKVSCKASGGTFGSYAISWVRQAPGQGLEWMGRIIPILGIATYAQKFQGRVTITADKSTSTAYMDLSSLRSEDTAVYYCARGKGEFEGMDVWGQGTTVT VSSSEQ ID NO: 25; heavy chain variable acceptor Acc.# IMGT# IGHV1-69-2*01EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATSEQ ID NO: 26; heavy chain variable acceptor Acc.# IMGT#IGKJ1*01QHWGQGTLVTVSSSEQ ID NO: 27; light chain variable acceptor Acc. # IMGT#IGKV2-30*02Acc. # IMGT#IGKV2-30*02DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPSEQ ID NO: 28; light chain variable acceptor Acc. # IMGT#IGKJ2*01YTFGQGTKLEIKSEQ ID NO: 29; Light chain variable acceptor Acc. # AAZ09048.1DVVMTQSPLSLTVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPGQSPRRLIYRVSHWDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTYWPLTFGQGTKLEIKSEQ ID NO: 30; Murine 3D6 VH nucleic acid sequence:GAGGTTCAGCTGCAGCAGTCTGGGGCTGACCTTGTGAGGCCAGGGGCCTTAGTCAAGTTGTCCTGCAAAGCTTCTGGCTTCAACATTAAAGACTACTATTTGCACTGGGTGAGGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGATGGATTGATCCTGAGAATGGTGATACTGTATATGACCCGAAGTTCCAGGGCAAGGCCACTATAACAGCAGACACATCCTCCAATACAGCCTACCTGCAGCTCGGCAGCCTGACATCTGAGGACACTGCCGTCTATTTCTGTTCTACCCTTGACTTCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCASEQ ID NO: 31; Murine 3D6 VL nucleic acid sequence:GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGTSEQ ID NO: 32; Murine CDR-H1 Kabat DYYLHSEQ ID NO: 33; Murine CDR-H1 Chothia GFNIKDYSEQ ID NO: 34; Murine CDR-H2 Chothia DPENGDSEQ ID NO: 35; Murine CDR-H2 AbM WIDPENGDTVSEQ ID NO: 36; Murine CDR-L1 Contact KTYLNWLSEQ ID NO: 37; Murine CDR-L2 Contact RLIYLVSKLDSEQ ID NO: 38; Murine CDR-L3 Contact WQGTHFPYSEQ ID NO: 39; Murine CDR-H1 Contact KDYYLHSEQ ID NO: 40; Murine CDR-H2 Contact WIGWIDPENGDTVSEQ ID NO: 41; Murine CDR-H3 Contact STLDSEQ ID NO: 42; Alternate Kabat-Chothia CDR-H1 GFTIKDYYLHSEQ ID NO: 43; Alternate Kabat CDR-H2 WIDPEDGDTVYAPKFQGSEQ ID NO: 44; consensus VH amino acid sequence from FIG. 2EVQLVQSGAEVVXPGALVKISCKASGFNIKDYYLHWVRQRPEQGLEWIGWIDPENGDTVYDPKFQGXATITADTSTNTAYLQLGSLTSEDTAVYFCSTLDFWGQGTLVTVSSSEQ ID NO: 45; consensus VL amino acid sequenceDVVMTQSPLSLSVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIKRSEQ ID NO: 46; hu3D6VHv1bA11B6G2:EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWVDPEDGDTVYAPKFQGRATITADTSTDTAYLELGSLTSEDTAVYFCSTLDFWGQGTLVTVSSSEQ ID NO: 47; hu3D6VHv1bA11B6H3:EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPEDGDTVYAPKFQGRATITADTSTDTAYLELGSLTSEDTAVYFCSTLDFWGQGTLVTVSSSEQ ID NO: 48; hu3D6VHv1c:EVQLVQSGAEVKRPGALVKISCKASGFNFKDYYLHWVRQRPEQGLEWMGWIDPENGDTVYDEKFQGRVTITADTSTNTAYLQLGSLTSEDTAVYFCSTLDFWGQGTLVTVSSSEQ ID NO: 49; hu3D6VHv1d:EVQLVQSGAEVKRPGALVKISCKASGYTFTDYYLHWVRQRPEQGLEWMGWVDPEDGDTVYAEKFQGRVTITADTSTNTAYLQLGSLTSEDTAVYFCSTLDFWGQGTLVTVSSSEQ ID NO: 50; hu3D6VHv1e:EVQLVQSGADVvkPGALVKISCKASGFTIKDYYLHWVRQRPEQGLEWIGWIDPENGDTVYAEKFQGRVTITADTSTNTAYLeLGSLTSEDTAVYFCSTLDFWGQGTTLTVSSSEQ ID NO: 51; hu3D6VHv1f:EVQLVQSGADVVKPGALVKISCKASGFTIKDYYLHWVRQRPGQGLEWIGWVDPEDGDTVYAEKFQGRVTITADTSTDTAYMELGSLTSEDTAVYFCSTLDYWGQGTTLTVSSSEQ ID NO: 52; hu3D6VHv3:EVQLVQSGAEVKKPGATVKISCKVSGFNIKDYYLHWVRQAPGKGLEWMGWIDPENGDTVYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCSTLDFWGQGTLVTVSSSEQ ID NO: 53; hu3D6VHv3b:EVQLVQSGAEVKKPGALVKISCKVSGYNFKDYYLHWVRQAPGKGLEWMGWIDPENGDTVYDEKFQGRVTITADTSTNTAYMELGSLRSEDTAVYYCSTLDFWGQGTLVTVSSSEQ ID NO: 54; hu3D6VHv3c:EVQLVQSGAEVKKPGALVKISCKVSGYTFTDYYLHWVRQAPGKGLEWMGWVDPEDGDTVYAEKFQGRVTITADTSTNTAYMELGSLRSEDTAVYYCSTLDFWGQGTLVTVSSSEQ ID NO: 55; hu3D6VHv4:EVQLVQSGAEVVKPGATVKISCKVSGFNIKDYYLHWVRQRPGKGLEWIGWIDPENGDTVYDPKFQGKATITADTSTNTAYLELGSLTSEDTAVYYCSTLDFWGQGTLVTVSSSEQ ID NO: 56; hu3D6VHv4b:EVQLVQSGAEVVKPGALVKISCKVSGYNFKDYYLHWVRQRPGKGLEWMGWIDPENGDTVYDEKFQGRVTITADTSTDTAYLELGSLTSEDTAVYYCSTLDFWGQGTLVTVSSSEQ ID NO: 57; hu3D6VHv4c:EVQLVQSGAEVVKPGALVKISCKVSGYTFTDYYLHWVRQRPGKGLEWMGWVDPEDGDTVYAEKFQGRVTITADTSTDTAYLELGSLTSEDTAVYYCSTLDFWGQGTLVTVSSSEQ ID NO: 58; Alternate Kabat-Chothia CDR-H1 (derived from hu3D6VH1c).GFNFKDYYLH SEQ ID NO: 59; Alternate Kabat-Chothia CDR-H1, (derived fromhu3D6VHv1d, hu3D6VHv3c, and hu3D6VHv4c). GYTFTDYYLHSEQ ID NO: 60; Alternate Kabat-Chothia CDR-H1 (derived fromhu3D6VHv3b and hu3D6VHv4b) GYNFKDYYLHSEQ ID NO: 61; Alternate Kabat CDR-H2 (derived from hu3D6VHv1bA11B6G2).WVDPEDGDTVYAPKFQGSEQ ID NO: 62, Alternate Kabat CDR-H2 (derived from hu3D6VHv1c,hu3D6VHv3b, AND hu3D6VHv4b. WIDPENGDTVYDEKFQGSEQ ID NO: 63; Alternate Kabat CDR-H2 derived from hu3D6VHv1d,hu3D6VHv1f, hu3D6VHv3c, and hu3D6VHv4c). WVDPEDGDTVYAEKFQGSEQ ID NO: 64; Alternate Kabat CDR-H2 (derived from hu3D6VHv1e).WIDPENGDTVYAEKFQGSEQ ID NO: 65; Alternate Kabat CDR-H3 (derived from hu3D6VHv1f) LDYSEQ ID NO: 66; heavy chain variable region of the mouse 6A10 antibody.EVQLQQSGAELVRSGASVKLSCTASGLNIKDYYIEWVKQRPEQGLEWIGWIDPENDDTEYAPKFQGRATLTTDTSSNTAYLQLSSLTSEDTAVYYCTPLDYWGQGTSVTVSSSEQ ID NO: 67; Kabat/Chothia composite CDR-H1 of the mouse 6A10 antibody.GLNIKDYYIH SEQ ID NO: 68; Kabat CDR-H2 of the mouse 6A10 antibody.WIDPENDDTEYAPKFQG SEQ ID NO: 69; Kabat CDR-H3 of the mouse 6A10 antibodyLDY SEQ ID NO: 70; Mus VH structure template (PDB#1CR9_H)KVKLQQSGAELVRSGASVKLSCTASGFNIKDYYIQWVKQRPEQGLEWIGWIDPENGNSEYAPRFQGKATMTADTLSNTAYLQLSSLTSEDTAVYYCNADLHDYWGQGTTLTVSSSEQ ID NO: 71; consensus VH amino acid sequence from FIGS. 4A and 4BEVQLVQSGAEVVKPGALVKISCKASGFNIKDYYLHWVRQRPGQGLEWIGWIDPENGDTVYDPKFQGRVTITADTSTNTAYLELGSLTSEDTAVYFCSTLDFWGQGTLVTVSSSEQ ID NO: 72; heavy chain of chimeric 3D6 antibodyEVQLQQSGADLVRPGALVKLSCKASGFNIKDYYLHWVRQRPEQGLEWIGWIDPENGDTVYDPKFQGKATITADTSSNTAYLQLGSLTSEDTAVYFCSTLDFWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 73; light chain of chimeric 3D6 antibodyDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

What is claimed is:
 1. A nucleic acid encoding a mature heavy chainvariable region fused to a heavy chain constant region, wherein themature heavy chain variable region has an amino acid sequence comprisingany one of SEQ ID NOS:15-19 and SEQ ID NOS:46-57.
 2. The nucleic acid ofclaim 1, wherein the mature heavy chain variable region has an aminoacid sequence comprising SEQ ID NO:15.
 3. A recombinant expressionvector comprising the nucleic acid of claim
 2. 4. A host celltransformed with the recombinant expression vector of claim
 3. 5. Thenucleic acid of claim 1, wherein the heavy chain constant region is amutant form of a natural human heavy chain constant region which hasreduced binding to a Fcγ receptor relative to the natural human heavychain constant region.
 6. The nucleic acid of claim 1, wherein the heavychain constant region is of IgG1 isotype.
 7. The nucleic acid of claim1, wherein the heavy chain constant region has at least one mutation. 8.The nucleic acid of claim 7, wherein the mutation reduces complementfixation or activation by the constant region.
 9. The nucleic acid ofclaim 8, having a mutation at one or more of positions 241, 264, 265,270, 296, 297, 318, 320, 322, 329 and 331 by EU numbering.
 10. Thenucleic acid of claim 9, wherein the heavy chain constant region hasalanine at positions 318, 320 and
 322. 11. The nucleic acid of claim 1,wherein the isotype of the constant region is human IgG2 or IgG4isotype.
 12. A recombinant expression vector comprising the nucleic acidof claim
 1. 13. A host cell transformed with the recombinant expressionvector of claim
 12. 14. A nucleic acid encoding a mature light chainvariable region, wherein the mature light chain variable region has anamino acid sequence comprising any one of SEQ ID NOS:_21-23.
 15. Arecombinant expression vector comprising the nucleic acid of claim 14.16. A host cell transformed with the recombinant expression vector ofclaim
 15. 17. A method of humanizing a mouse antibody, the methodcomprising: (a) selecting human acceptor antibody sequences; (b)identifying CDRs and variable region framework amino acid residues ofthe mouse antibody to be retained; (c) determining amino acid sequencesof a humanized heavy chain variable region and a humanized light chainvariable region from the CDRs and variable region framework amino acidresidues of the mouse antibody to be retained and the one or more humanacceptor antibody sequences; (c) synthesizing a nucleic acid encodingthe humanized heavy chain and a nucleic acid encoding the humanizedlight chain; and (d) expressing the nucleic acids in a host cell toproduce a humanized antibody; wherein the mouse antibody ischaracterized by a mature heavy chain variable region of SEQ ID NO:7 anda mature light chain variable region of SEQ ID NO:11.